WO2000046360A1

WO2000046360A1 - Improved helper dependent vector system for gene therapy

Info

Publication number: WO2000046360A1
Application number: PCT/US2000/002405
Authority: WO
Inventors: Andrew Bett; Volker Sandig; Rima Youil
Original assignee: Merck & Co., Inc.
Priority date: 1999-02-04
Filing date: 2000-01-31
Publication date: 2000-08-10
Also published as: CA2360796A1; EP1151091A1; EP1151091A4; JP2002535986A

Abstract

The present invention features helper-dependent adenoviral vector elements, and helper adenoviral elements, that enhance the production and isolation of helper-dependent adenoviral vectors. Such elements include a modified packaging signal having low homology to, and preferably less activity than, a wild-type packaging signal, an E4 non-coding segment directly joined to the 5' ITR that confers a selective advantage, and stuffer region(s) that provide a helper-dependent adenoviral vector with a GC content of about 50 % to about 60 %. The modified packaging signal is preferably used in a helper virus to decrease recombination and generation of the virus. The E4 non-coding segment and the stuffer region(s) are preferably used in a helper-dependent adenoviral vector to provide the vector with a growth advantage over a helper virus.

Description

TITLE OF THE INVENTION

IMPROVED HELPER DEPENDENT VECTOR SYSTEM FOR GENE THERAPY

FIELD OF THE INVENTION

This invention relates to adenoviral vectors which are useful for nucleic acid delivery into a cell such as in gene delivery and nucleotide vaccine applications. It also relates to host cells and methods using these vectors.

BACKGROUND OF THE INVENTION

The references cited herein are not admitted to be prior art to the claimed invention.

Adenoviral vectors provide a vehicle for introducing nucleic acids into a cell in vitro and in vivo. Over 100 different serotypes, of which 47 are of human origin, have been reported. (Hierholzer, et al., J. infect. Dis. 759:804-813, 1988.) Adenoviruses type 2 and 5 used to produce adenoviral vectors are well characterized.

A human adenovirus contains a 5' inverted terminal repeat (ITR); a packaging signal; region El made up of the early regions El A and E1B; region E2; region E3, region E4; the late regions L1-L5; and a 3' ITR. Regions El and E4 contain regulatory proteins, region E2 encodes proteins required for replication, and the L region encodes for the structural proteins of the virus. The E3 region is dispensable for virus growth in vitro. (Hitt, et al, Advances in Pharmacology 40: 137-206, 1996.)

A replicating viral vector based on the adenovirus must contain adenovirus cis elements needed for replication. Packaging also requires cis elements on the vector whereas the necessary proteins can be supplied in trans. In trans supplementation can be brought about by specialized cells and/or additional viruses producing the needed proteins. (Hitt, et al., Advances in Pharmacology 40:131-206, 1996.) Different types of adenoviral vectors have been developed including those lacking one or more components of the adenoviral genome. An adenoviral vector produced without components of the adenoviral genome provides advantages such as in increasing the amount of foreign nucleic acid that can be included with the adenoviral vector and removing adenoviral genes whose expression can have detrimental effects. (Hitt, et al., Advances in Pharmacology 40:131-206, 1996.) Adenoviral vectors lacking all viral protein-coding sequences have been developed. These vectors require supplementation of viral regulatory and structural proteins supplied in trans for packaging and rescue. A second adenovirus carrying genes necessary for virus growth can be used to provide in trans the required supplementation of proteins. (Mitani, et al, Proc. Natl. Acad. Sci. USA 92:3854- 3858, 1995; Fisher, et al, Virology 277.T 1-22, 1996; Kochanek, et al., Proc. Natl. Acad. Sci. USA 95:5731-5736, 1996; Parks, et al., Proc. Natl. Acad. Sci. USA 95: 13565-13570, 1996; Parks, et al, J. Virology 77(N):3293-3298, 1997; and Schiedner, et al., Nature Genetics 18: 180-183, 1998.)

SUMMARY OF THE INVENTION

The present invention features helper-dependent adenoviral vector elements, and helper adenoviral elements, that enhance the production and isolation of helper-dependent adenoviral vectors. Such elements include a modified packaging signal having low homology to, and preferably less activity than, a wild-type packaging signal, an E4 non-coding segment directly joined to the 5' ITR that confers a selective advantage, and stuffer region(s) that provide a helper-dependent adenoviral vector with a GC content of about 50% to about 60%. The modified packaging signal is preferably used in a helper virus to decrease recombination and generation of the virus. The E4 non-coding segment and the stuffer region(s) are preferably used in a helper-dependent adenoviral vector to provide the vector with a growth advantage over a helper virus.

A "helper-dependent adenoviral vector" refers to a viral vector containing the cis elements needed for adenovirus replication and packaging (also referred to herein as viral generation), but lacking the necessary trans elements for adenovirus replication and packaging. The cis elements needed for viral generation is an adenovirus 3' ITR, a packaging signal, and an adenovirus 5' IRT. Trans elements needed for viral generation are the proteins encoded by the El, E2, and E4 and L adenovirus regions. Preferably, the helper-dependent adenoviral vector lacks nucleic acid encoding for any adenovirus proteins. A helper-dependent adenoviral vector is particularly useful as a vector for delivering genes into cells in vivo.

A "helper virus" refers to a virus expressing one or more proteins needed for viral generation of a helper-dependent adenovirus. Preferably, the helper virus contains the adenovirus 3' IRT, an adenovirus packaging signal, an adenovirus 5' IRT, and encodes for the proteins of the Adenovirus E2 and E4 and L regions. Trans elements not provided for by a helper virus may be provided for by other means such as by a cell. For example, a 293 cell can be employed to supply in trans needed El proteins in conjunction with a helper virus not expressing such proteins. Helper-dependent adenoviral vectors can be produced by co- cultivating with helper viruses in cell lines, wherein the helper virus alone or in combination with the cell line provide the trans functions needed for adenovirus generation. During co-cultivation of a helper-dependent adenoviral vector and a helper virus the two compete for packaging, producing a mixed viral population. Often, during co-cultivation the helper virus has a selection advantage, such that the mixed population of helper virus and helper-dependent adenoviral vector becomes predominantly helper virus, rather than the desired helper-dependent adenoviral vector. Even when this phenomenon does not occur, the presence of lesser, but significant, amounts of helper viruses still contaminates the helper- dependent adenoviral vector preparation. Therefore, the viral populations may have to be separated physically and/or production of helper particles suppressed for the helper-dependent adenoviral vector to be used. Preferably, the percentage of helper virus in a purified helper-dependent adenoviral vector stock is below about 0.5%. Thus, a first aspect of the present invention describes a nucleic acid molecule comprising an excisable low homology packaging signal cassette. The packaging cassette comprises a low homology packaging signal cassette flanked by a recombinase recognition sequence. The modified packaging signal has low homology to a wild-type adenovirus packaging signal.

"Flanked by a recombinase recognition sequence" indicates the presence of a recombinase recognition sequence 3' and 5' of the packaging signal allowing for excision of the flanked nucleic acid containing the packaging signal by a recombinase recognizing the recognition sequence. The recombinase recognition sequence does not need to be immediately 3' or 5' of the modified packaging signal. Preferably, the 3' and 5' recognition sequence are about 200 bp to about 2,000 bp apart.

The modified packaging signal has low homology to a wild-type adenovirus packaging signal and still allows for packaging of the helper virus during its separate production. Preferably, the modified packaging signal is less efficient than a wild-type adenovirus packaging signal. "Low homology" refers to a maximum of about 25 bp of contiguous sequence homology (100% sequence identity) between the modified packaging signal and a wild type packaging signal present in an adenovirus, preferably human adenovirus serotype 5. Low homology is determined by aligning the modified packaging signal with wild type packaging signal to obtain a stretch of maximum contiguous homology with sequences present in both the modified and wild type packaging signals. Preferably, contiguous sequence homology is at most 23 bp.

"Less efficient" than a wild-type adenovirus packaging signal indicates that the helper virus has a lower level of packaging when it contains the modified packaging signal than it has when it contains a wild-type packaging signal present in an adenovirus, preferably from a human adenovirus group C serotype, more preferably serotype 5. Preferably, the helper virus can still be produced at yields of at least 300 PFU/cell in cells lacking a compatible recombinase. Efficiency is preferably determined using titration by end point dilution assay described in the Examples below.

Reference to "modified" is not a limitation as to how the packaging signal sequence is produced. Instead, "modified" indicates that the sequence is not a wild-type sequence.

Another aspect of the present invention describes an adenoviral helper virus for production of helper-dependent vectors comprising:

(a) an adenovirus genome having an El region deletion;

(b) an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP site, a modified packaging signal and a 3' loxP site, wherein the modified packaging signal has low homology to and is less efficient than the wild-type packaging signal; and

(c) an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region.

Another aspect of the present invention describes a helper-dependent adenovirus vector comprising the following elements (in 5' to 3' order): (a) a 5'- in verted terminal repeat sequence; (b) a packaging signal; (c) one or more heterologous gene expression cassettes; (d) an optionally present E4 non-coding segment conferring a selective advantage; and (e) a 3' ITR; wherein (d) and (e) are located in the distal 400 bp of the adenovirus genome, and wherein the only adenoviral sequences present in the vector are said 5 -inverted terminal repeat, said 3'- inverted terminal repeat, said packaging signal, and said nonexpressed segment of the E4 region.

The "E4 non-coding segment" refers to the nonexpressed segment of the E4 region co-localized with the E4 promoter. The segment is adjacent to the 3' ITR and, in human adenovirus serotype 5, is about 300 bp in length.

Another aspect of the present invention describes a helper-dependent adenovirus vector having a GC content between about 50% and about 60%. The vector comprises in a 5' to 3' direction: (a) a 5' ITR; (b) a packaging signal cassette directly joined to the 3' of the 5' ITR; (c) a first stuffer DNA at least about 1 kb; (d) at least one heterologous expression cassette; (e) a second stuffer DNA at least about 1 kb; (f) an optionally present non-coding E4 segment; and (g) a 3' ITR. Element (f) if present is directly joined to element (g). The virus does not encode one or more adenovirus proteins needed for adenovirus generation and is about 28 kb to about 36 kb. Preferably, the virus does not encode any adenovirus proteins.

"Directly joined" indicates the absence of significant intervening sequences (e.g., less than about 150 bp) that interfere with the function of "joined" groups.

"Stuffer sequences" refer to nucleic acid regions that do not express protein when present in a helper-dependent adenoviral vector. Preferably, such regions are mammalian non-gene regions or introns.

Another aspect of the present invention describes a cell line infected with a helper virus, a helper-dependent viral vector, or both the virus and the vector. Preferably, the cell line expresses El proteins and a recombinase, and is infected with a helper virus that (1) contains an excisable low homology signal packaging cassette, and (2) does not express El proteins needed for viral generation.

Another aspect of this invention is a method of generating helper- dependent adenoviral gene vectors in a cell line expressing El proteins and Cre recombinase comprising: a) infecting the cell line with a helper-dependent vector comprising: a

5' ITR, a packaging signal, at least one heterologous expression cassette, human genomic stuffer DNA and a 3' ITR, wherein the overall size of the helper-dependent vector is between about 28 kb and 36 kb, and wherein no functional adenoviral coding sequences and no bacterial origin of replication or bacterial marker genes are present; b) infecting the cell line with a helper virus comprising: an adenovirus genome having an El region deletion; an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP site, a modified packaging signal and a 3' loxP site, wherein the modified packaging signal has low homology to and is less efficient than the wild- type packaging signal; and an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region; and c) obtaining the generated helper-dependent viral vectors. Another aspect of the present invention describes a method of generating helper-dependent adenoviral vectors. The method involves producing a cell comprising: (i) trans functions needed for adenovirus generation and (ii) a helper-dependent adenoviral vector comprising the necessary cis functions needed for adenovirus generation and at least one heterologous expression cassette, wherein the helper-dependent adenoviral vector does not encode for any adenovirus proteins, is about 28 kb to about 36 kb, and has a GC content between about 50% and about 60%. The cell is used to generate the helper-dependent vector. Reference to a cell comprising the trans functions needed for adenovirus generation indicates such functions are present in the cell, but need not be part of the cellular genome. The trans functions can be provided, for example, by a helper virus, the cellular genome, or a combination of both. Another example of a source of trans functions is a plasmid. Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows the shuffling of fragments of a stuffer to prevent genomic integration of the expression units. FIGURE 2 is a gel showing restriction digests of helper-dependent adenoviral vector DNA after competitive rescues of: a) stkl20gfp-E4-promoter and stkl20gfp; b) stkl20gfp-E4 and stkl20gfp; and c) stkl20gfp-E4-promoter and stkl20gfp-E4. FIGURE 3 is a gel showing restriction digests of helper-dependent adenoviral vectors grown with the helper AdLCδBHG and a helper of this invention.

FIGURE 4 shows the structure of different helper-dependent adenoviral vectors.

FIGURE 5 shows autoradiographs of labeled restriction fragments from helper-dependent adenoviral vectors. The figure illustrates the results of competition experiments involving groups of helper-dependent adenoviral vectors from transfection to passage 6. "A" is a competition of backbones containing the shuffled fragments of cosmid HUMDXS455A (C4). "B" is a competition of backbones containing part of the HPRT (hypoxanthine guanine phosphoribosyltransferase) genomic gene. "C" is a competition between 2 backbones one containing part of the HPRT genomic gene the other containing the shuffled fragments of HUMDXS455A (C4).

FIGURE 6 is a graph showing the correlation between GC content and helper virus contamination. FIGURE 7 is a graph showing the balance between helper and helper- dependent adenoviral vectors at different time points after infection. The figure demonstrates the replication potential of different helper-dependent adenoviral vectors with the mouse Erytropoetin gene relative to the helper virus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is particularly useful for obtaining and isolating helper-dependent adenoviral vectors produced using a helper virus. Co-cultivation of a helper-dependent adenoviral vector and a helper virus produces two different particles competing for packaging and results in a mixed viral population. The production of the helper virus in the mixed viral population reduces the amount of helper-dependent adenoviral vector produced, provides a viral contaminant that interferes with applications of the helper-dependent adenoviral vector, and produces a source of nucleic acid that can be recombined with helper-dependent adenoviral vectors As described herein, production and isolation of helper-dependent adenoviral vectors can be facilitated in different ways. One way is to employ helper- dependent adenoviral vectors containing elements favoring viral generation of the helper-dependent adenoviral vector over a co-cultured helper virus. Another way is to employ helper viruses containing elements that can be employed to reduce the ability of the helper virus to grow, and /or recombine with the helper-dependent adenoviral vector.

The term "vector" employed herein is used in its broadest sense, and is meant to encompass a linear virus which can carry a heterologous gene, and or a plasmid which can contain one or more regions of a virus genome. The term encompasses a helper-dependent adenoviral vector and a helper virus.

Adenovirus components present in a helper or a helper-dependent vector may in general be provided from any adenovirus. Preferably, a human adenovirus serotype is used; more preferably, human adenovirus serotype 1-47; and more preferably, a group C serotype (e.g., serotypes 1, 2, 5, and 6). Preferred group C serotypes are human adenovirus serotype 2 or 5, and more preferably serotype 5.

A helper-dependent adenoviral vector preferably contains a heterologous expression cassette. Such a cassette is made up of a heterologous gene functional coupled to a promoter and regulator elements needed for gene expression, and is particularly useful for introducing nucleic acid into a cell. The introduced nucleic acid preferably encodes for a gene or is able to regulate gene expression. Nucleic acid able to regulate gene expression include ribozymes and antisense nucleic acids.

The ability to introduce nucleic acid into a cell using the present invention has different applications such as in gene therapy, in antibody production (e.g., a vaccine) and research to examine the regulation of a gene in vivo or in vitro. Different genes can be introduced into a cell such as Factor VIII, Factor VIX, CFTR, OTC, LDL, VEGF, FGF, EPO, HSV-TK, EL-2, IL-12, p53, HLA-B7, -interferon, and cytosine deaminase. A promoter is a DNA sequence directing the synthesis of RNA through an RNA polymerase. Suitable promoters depend upon the gene and intended use, and may include promoters such as EFl , CMV, chicken α-actin (Arnold, et al., Nucleic Acids Res 1988 Mar 25;16(6):2411-29), and muscle creatine kinase (Johnson, et al, Mol Cell Biol 1989 Aug;9(8):3393-9 ). Generally, the regulatory elements that are present include a πbosome binding site, a terminator, and an optionally present operatoi. Another preferred element is a polyadenylation signal. Regulatory systems are available to control gene expression, such as GENE-SWITCH™ (Wang, et al, Gene Ther. 1997 May;4(5):432- 41, U.S. Patent No. 5,874,534 and International Publication WO 93/23431, each of which are hereby incorporated by reference herein) and those involving the tetracychne operator (U.S. Patent Nos 5,464,758 and 5,650,298, both of which are hereby incorporated by reference herein).

A helper-dependent adenoviral vector is preferably from about 28 kb to about 36 kb to provide for efficient packaging. An adenovirus can accommodate up to about 105% of the wild-type genome and has a lower packaging limit of about 75% of the wild-type genome. (See, Parks, et al, J. Virology 71(4)3293-3298, 1997, hereby incorporated by reference herein.) In a preferred embodiment, there are no bacteπal plasmid-based sequences (such as an oπgin of replication or bacteπal marker genes) present in the helper-dependent adenoviral vector.

Construction of helper viruses and helper-dependent viruses can be achieved based on the guidance provided herein using techniques well known in the art, such as those descπbed by Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al, in Molecular Cloning, A laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989, both of which are hereby incorporated by reference herein Additional descriptions of useful techniques are provided, for example, in the Example section provided below and references such as Mitani, et al, Proc. Natl. Acad. Sci. USA 92:3854-3858, 1995; Fisher, et al, Virology 277:11-22, 1996; Kochanek, et al , Proc. Natl Acad. Sci. USA 93. 5731- 5736, 1996; Parks, et al, Proc. Natl Acad. Sci. USA 95.13565-13570, 1996; Parks, et al, J. Virology 71(4):3293-3298, 1997; and Schiedner, et al, Nature Genetics 18: 180-183, 1998; each of which are hereby incorporated by reference herein.

The helper-dependent adenoviral vector and the helper virus can be generated in a wide range of host cells able to be infected by adenovirus. Examples of host cells include 293 cells, 911 cells and PERC.6 cells (WO 97/00326 hereby incorporated by reference herein). The employed cell can be constructed to provide for useful proteins such as viral proteins or a recombinase.

HELPER VIRUS Helper virus elements useful for facilitating the production and isolation of helper-dependent viruses include a low homology excisable packaging signal cassette and a substituted E3 region. Preferred helper viruses contain both a low homology excisable packaging signal and a substituted E3 gene.

Low Homology Excisable Packaging Signal Cassette

The low homology excisable packaging signal cassette contains a modified packaging sequence which fulfills the role of an adenovirus packaging signal, has a low homology to the Adenovirus packaging signal, and is flanked by a 3' and 5' recombinase recognition sequence. Preferably, the modified packaging signal is less efficient than a wild type packaging signal. Advantages of such low homology excisable packaging signals include: (1) less recombination between the helper virus and helper-dependent adenoviral vector; and (2) when a less efficient packaging signal is used, less packaging of those helper viruses which escaped excision of the packaging signal.

Recombinase recognition sequences recombine when acted on by a recombinase that recognizes the sequences, resulting in the excision and circulaπsation of intervening nucleic acid. The recognition sequences should be sufficiently far apart, at least about 180 bp, to allow for the proper positioning of nucleic acid segments being recombined. Preferably, the recombinase recognition sequence is loxP or frt.

The loxP recognition sites can be positioned 3' and 5' of the packaging signal to allow for excision by Cre recombinase. Cells, such as 293 ere cells, stably expressing Cre recombinase can efficiently remove nucleic acid located between loxP recognition sites. (See, Parks, et al, Proc. Natl Acad. Sci. 95:13565-13570, 1996 and Parks, et al, J. Virology 71 (4 ):3293-329S, 1997, both hereby incorporated by reference herein).

The frt recognition sites can be positioned 3' and 5' of the packaging signal to allow for excision by Flp recombinase. Cells stably expressing Flp recombinase can efficiently remove nucleic acid located between frt recognition sites. Flp-mediated gene modifications are descπbed in U.S. Patent No. 5,564,182, hereby incorporated by reference herein.

Homology between a helper and the helper-dependent adenoviral vector encourages recombination events between the two, resulting m unwanted changes in the structure of the helper-dependent adenoviral vector or the helper virus, and leading to an increased contamination by helper virus. For example, if the first of a loxP site flanking the packaging signal is removed by homologous recombination within the packaging signal, the resulting helper virus escapes selection in 293cre cells because the Cre recombinase is no longer able to excise the packaging signal. Sequences for different low homology excisable packaging signal cassettes can readily be designed taking into account recombinase recognition sequences and adenovirus wild-type packaging signal sequences using the guidance provided herein. The provided guidance focuses on the adenovirus serotype 5 packaging signal to illustrate the production of a modified packaging signal. Such guidance is applicable to producing other modified packaging signals taking into account other adenovirus serotypes.

The wild-type packaging signal of adenovirus serotype 5 is formed by at least seven functional units called A repeats, which are located between nt 230 and nt 380 of the genome. The A elements have the consensus sequence ATTTGNsGC

(Schmid et al, 1997 J. Virol. 71:3375-3384, which is hereby incorporated by reference). The modified packaging signal of this invention preferably comprises less packaging elements than the wild-type (which has seven elements), preferably from about two to six elements, and more preferably from three to five elements. In a preferred embodiment, the modified packaging signal contains only four out of the seven original packaging elements (elements Al to AIV). The four elements are preferably in a modified form with two strong elements (Al and AH) present. The position of the elements relative to each other may be changed, but in preferred embodiments, it is maintained. In order to reduce contiguous sequence homology, the eight ambiguous nucleotides of the consensus sequence (ATTTGNsGC; SEQ. ID. NO. 1) within each

A element are preferably replaced by sequences taken from a different A element. For example, the eight nucleotides within Al were replaced by those from AV; and the eight nucleotides within AH were replaced by those from AVI. In addition, a new element was created between Au and AIII starting 21 bp after AH, by changing the existing nucleotides to the consensus sequence. Two more nucleotides were exchanged within AIV: ATTTTGTGTT (SEQ. ID. NO. 2) was changed to ATTTTGTTGT (SEQ. ID. NO. 3). One embodiment of a synthetic packaging signal is given in SEQ. ID. NO. 4. Desired nucleic acid sequences can be produced using different techniques including those involving the creation of nucleic acid mutations and nucleic acid synthesis techniques. Examples of such techniques are provided in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al, in Molecular Cloning, A laboratory Manual, 2^nd Edition, Cold Spπng Harbor Laboratory Press, 1989, both of which are hereby incorporated by reference herein.

E3 Insertion Homologous recombination between viral sequences (El) present in the cell lines used for propagation of either helper virus (lacking El) or helper- dependent adenoviral vector, and the helper virus itself, has resulted in the generation of wild-type viruses. Homologous recombination results in the insertion of the El region into an El-deleted virus genome. To avoid this problem, in preferred embodiments of this inventions, the helper virus also contains an insertion element. Preferably, the insertion element does not contain a promoter or encode for a protein when present in the helper virus. More preferably, the insertion element is non- coding repeat-free human DNA devoid of splice signals.

Parks, et al, Proc. Natl. Acad. Sci. USA 95:13565-13570, 1996, descπbe the use of an E3 region containing an insert encoding for the ampicillin gene, the bacteπal oπgm of replication, a CMV promoter and the luciferase gene (AdLC8BHG101uc). The insert protected against the production of viable viruses having a wild type El produced by homologous recombination. However, the insert impairs virus generation, most likely by interference with fiber gene expression. The resulting level of viral generation is disfavored for the production of viral stock. The use of non-cod g sequences as an E3 insertion element, preferably human non-gene sequences, provides for more efficient viral generation than that observed with E3 msertional elements encoding for bacteπal protein described by Parks, et al, Proc. Natl. Acad. Sci. USA 95.13565-13570, 1996 (see Table 1, infra.) The use of intron sequences are prefeπed because such sequences do not contain hidden splice signals that could interfere with correct splicing of the fiber message.

The size of the insertion element (preferably between about 2800 and 3500 bp, more preferably about 2900 bp) is chosen to avoid the generation of potentially harmful wild-type viruses while maintaining a virus size close to wild-type size. Starting with a helper virus having a deleted El, if homologous recombination occurs resulting in insertion of the El region, the recombined adenoviral vector which also contains the E3 insertion element would have a genome size of approximately 38350 bp (108.1% of wild-type size). Because this size exceeds the packaging limit of Adenovirus 5, no viable virus would be produced.

The insertion element may be from virtually any source. In a preferred embodiment, a 2900 bp insert was taken from human chromosome 1 lql3, starting 13340 bp upstream of STS marker US 1337 (available from Genethon) and inserted into the Xba I site at position (nt) 28593 (referring to Ad5 wild-type).

The insertion element and its insertion point in E3 should be chosen to avoid any reduction in synthesis of the fiber protein. Such a reduction is likely to occur if the E3 region is modified because synthesis of fiber protein is dependent on coπect splicing of RNA from signals located in this region. We demonstrate that viruses containing the insert grow to titers similar to those obtained with viruses containing an unchanged E3 region.

HELPER-DEPENDENT ADENOVIRAL VECTOR Helper-dependent adenoviral vector elements useful for facilitating the production and stability of helper-dependent adenoviral vectors include an E4 non- coding segment that confers a selective advantage to a virus and stuffer region(s) that provide a helper-dependent adenoviral vector with a GC content of about 50% to about 60%. Preferably, the helper-dependent adenoviral vector contains an E4 non- coding segment conferring a selective advantage to the vector; and stuffer region(s) providing the vector with a GC content of about 50% to about 60% and an overall size of about 28 kb to about 36 kb. In a prefeπed embodiment, there are no bacterial plasmid-based sequences (such as an origin of replication or bacterial marker genes) present in the helper-dependent adenoviral vector.

Stuffer DNA

Stuffer sequences are used to provide a helper-dependent adenoviral vector with an overall size of about 28 kb to about 36 kb. A helper-dependent adenoviral vector containing only a heterologous expression cassette, adenoviral ITR's and a packaging signal may be quite small, in general about 5-10 kb, depending on the size of the heterologous expression cassette. Because such a small virus does not package efficiently stuffer sequences are added to provide a final size of about 28 kb to about 36 kb. Preferably, the stuffer provides the vector with a GC content of about 50% to about 60%. Stuffer sequences used in gene therapy applications should not contain active genes or be recognized as foreign in a human cell. A preferred stuffer sequence replicates as well as the adenovirus itself, is not recognized by the host as foreign DNA and does not lead to chromosomal integration of the transgene.

Genomic DNA of mammalian origin, or even more preferably of human origin, are preferred stuffer sequences for gene therapy applications to avoid eliciting an immune response. However, stuffer based on human genomic DNA is identical to chromosomal sequences in a human target cell and could allow for insertion of the heterologous expression cassette into the host genome by homologous recombination. This phenomenon could have a potentially dangerous effect and should be prevented.

To prevent integration of the heterologous expression cassette into the host genome, the contiguous stuffer sequence can be inteπupted, the individual fragments reversed and the expression units inserted at the specific breakpoints. (See for example, Figure 1.) Using this strategy, the transgene is not flanked by regions which would support homologous recombination. Additionally, potentially unstable genetic elements such as retrovirus LTR as well as genomic repeats (e.g., MIR, and ALU) should avoided or removed to prevent reaπangements during amplification.

Another consideration is the GC content of the virus. The GC content that stuffer sequences provide to a helper-dependent adenoviral vector has a major impact on yield and purity of the virus. Competition experiments between different viruses as well as individual analysis revealed a correlation between the GC content of a virus and growth properties. The GC content is preferably between about 50% and about 60% and even more preferably between 52% and 57% which is close to the GC of wild-type adenovirus. Without being limited to any particular theory, advantageous growth properties are believed to be based on more efficient generation of a virus with a higher GC content.

Preferably, stuffer sequences are provided such that the heterologous gene cassette(s) that are present are at least about 1.0 kb from the helper-dependent viral vector 3' end and 5' end, more preferably at least about 2.0 kb, and more preferably at least about 4.0 kb. Additionally, high AT rich segments such as those provided in the hprt fragment (59% AT) may be unfavorable even with a viral vector having an overall high GC content. A "high" AT is above about 55% AT.

E4 Non-coding Segment

The E4 region of adenovirus type 5 occupies about 3000 bp at the right end of the genome. Transcription from the E4 promoter is directed from the right end toward the center. Seven polypeptides are produced from open reading frames (ORFs) located within the region with the first ORF starting at nucleotide (nt) 406 from the right end of the virus.

It was observed that the E4 region is sometimes transferred from a helper to a helper-dependent adenoviral vector as a result of homologous recombination. This recombined helper-dependent adenoviral vector generally is favored in co-cultivation with the non-recombined helper-dependent adenoviral vector and dominates in vector preparations. Despite its strength, this recombined helper-dependent adenoviral vector cannot be used as a helper-dependent vector for gene therapy because it contains functional E4 genes. Expression of those E4 genes in a host cause an immune rejection of vector infected cells by the host.

In accordance with this invention, it has been determined that a noncoding segment of the E4 region confers a selective advantage when a virus is co- cultivated with a virus lacking the same element. The entire E4 region is not needed.

From adenovirus serotype 5, the part of E4 between the 3' ITR (starting 102 nt from the right end) and nt 405 which does not contain coding sequence is the segment that should be preferably included into the helper-dependent vector and is a non-coding E4 segment. E4 sequences located left of this element have no additional impact on virus strength. This is of importance because all genes (open reading frames, orfs) are located in the left part of the E4 region. Thus, another aspect of this invention is a ^'s-acting sequence located next to the 3' ITR (right virus end) which is preferably incorporated into the helper-dependent adenoviral vector because it supports amplification of the helper-dependent adenoviral vector.

Competition experiments were designed to determine the location of the beneficial element. Helper-dependent adenoviral vectors based on the vector stkl20 (which contains only a 5' ITR, a 3' ITR and packaging signal as its viral-based nucleotides, as described in Morsy et al, 1998, Proc. Natl Acad. Sci. USA 95:7866- 7871, which is hereby incorporated by reference) were constructed containing either the 3' ITR only (stkl20gfp), the 3' ITR and noncoding sequence of the E4 region until nt 400 from the right end (stkl20gfp-E4promoter ) or the 3' ITR and the complete E4 region to nt 3115 from the right end (stkl20gfp-E4). Pairs of these vectors were cotransfected at a 1:1 ratio to originate rescues of two competing helper-dependent adenoviral vectors.

After passage seven, viruses from at least three independent rescue procedures were purified, and DNA was extracted and analyzed by radioactively labeled restriction digests. The two competing helper-dependent adenoviral vectors have an almost identical restriction pattern. The only difference is in the size of one fragment, called the discriminating fragment. The discriminating fragment was found 5-10 fold stronger for stkl20gfp-E4-promoter than for stkl20gfp, and at least 4 fold stronger for stkl20gfp-E4 than for stkl20gfp (see Figures 2a and 2b). However, the discriminating fragments are of equal strength for stkl20gfp-E4-promoter and stkl20gfp-E4 in each rescue (see Figure 2c). Therefore an E4 element supporting helper-dependent adenoviral vector growth is present in stkl20gfp-E4 promoter and stkl20gfp-E4 and must be entirely located in the right part of the E4 region (between nucleotide -400 from the right end and the 3' ITR).

While not wishing to be bound by theory, it appears that there are two possible explanations for the selective advantage to the E4 segment-containing adenoviral vector: the E4 element allows for more effective replication of the virus or it confers improved packaging and stability. Our results indicate that the E4 segment does not effect replication. If infection is started with equal amounts of helper and helper-dependent adenoviral vectors, the ratio between helper and helper-dependent genomes inside the cell at the end of the production cycle (48 hours) is identical. However, 3-fold more helper-dependent adenoviral vectors were packaged for the genome containing the E4 segment compared to contaminating helper genomes. Thus, it appears that the E4 segment supports effective packaging.

EXAMPLES

Examples are provided below to further illustrate different features and advantages of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention. EXAMPLE 1

General Materials And Methods

Construction Of Plasmids Cloning techniques described in J Sambrook, E.F. Fritsch and T.

Maniatis, 1989 Molecular Cloning, Cold Spring Harbor Laboratory Press, which is hereby incorporated by reference, were used.

To generate plasmids containing the entire Ad5 genome, homologous recombination in E. coli based on the recF pathway was used. Competent cells of E. coli strain BJ 5183 were transformed with a plasmid vector containing the adenovirus genome and a fragment from the shuttle vector. This fragment contains the sequence to be inserted flanked on both sides by sequences homologous to the vector. In order to select against re-transformation of the vector without modification, it was linearized close to the insertion point. DNA isolated from individual colonies of BJ5183 was retransformed into the strain XL2 (Stratagene) and high yield plasmid preparations were obtained. Correct clones were determined by restriction analysis and sequencing.

Virus Rescue lOμg helper plasmid was digested with Pad and transfected by calcium phosphate coprecipitation into a 6 cm dish of subconfluent 293 cells. Directly after transfection cells were overlaid with αMEM/ 10% FCS/ 0.8 % Seaplaque agarose. 20-100 plaques appeared after 7-9 days. Plaques were taken 12 days after transfection and used to reinfect cells. After two intermediate passages the cell lysate was used to infect NUNC-cell factories (NUNC). Cells were harvested 48 hours after infection and virus was released by three freeze/thaw steps steps (-70°C /37°C). After treatment of the cleared lysate with BENZONASE nuclease, viruses were purified by ultracentrifugation on a CsCl gradient. After one step gradient (q= 1.5 / 1.35 1.25 g/cm3) the viruses were loaded on a continuous gradient formed from a CsCl solution of q= 1.35 g/cm3. Viruses were dialyzed against lOmM Tris pH 8.0, lOmM MgCl2 and 10% glycerol and stored in aliquots at -70°C. Helper-dependent adenoviral vectors were purified using substantially the same procedure. For analytical purposes virus obtained from two 15 cm dishes was purified. Virus DNA was cut with Hind III or other restriction endonucleases, restriction fragments were labeled using ocP33dATP or dCTP and Klenow polymerase and separated in 0.7% agarose gels. The gel was dried and exposed to film.

Determination Of Particle Titer lOμl of virus suspension was added to 90μl PBS/ 0.1% SDS and incubated at 50°C for 20 minutes. OD260 is determined spectrophotometri caliy and the concentration of virus particles was calculated based on the equation 1 OD26O = 1.1x1012 particles.

Extraction Of Viral DNA and Restriction Digest lOOμl of virus suspension were added to lOOμl lysis solution (lOmM Tris-HCl pH 7.5, lOmM EDTA, 0.5% SDS, 0.05% pronase) under constant vortex. After an incubation at 37°C for at least 2 hours followed by addition of 100/xl TE the lysate was extracted lx with Tris-HCl saturated phenol. Virus DNA was precipitated with two volumes of ethanol, washed extensively with 70% ethanol and resuspended in TE. Virus DNA was cut with Hind HI or other restriction endonucleases, restriction fragments were labeled using P33dATP or dCTP and Klenow polymerase and separated in 0.7% agarose gels. The gel was dried and exposed to film.

Extraction of Cellular DNA From Infected Cells

10⁶ cells are lysed inlOmM Tris-HCl pH 7.5, lOmM EDTA, 0.5% SDS and treated with 100μg/ml Proteinase K overnight at 58°C. The lysate was extracted with Tris saturated phenol, phenol/chloroform/ isoamylalcohol (24:24: 1) and chloroform, precipitated with two volumes of ethanol, washed extensively with 70% ethanol and resuspended in TE.

End Point Dilution Assay

96 well plates were seeded with 1x104 293 cells in lOOμl per well 24 hours before the assay. Each virus stock was diluted 103 to 108- fold. Dilutions were used to infect 24 wells each (50μl/ well). Positive wells were scored by cytopatic effect after 14 days and the virus titer was calculated based on two dilutions with intermediate numbers of positive wells taking the dilution factor into account.

Quantitative PCR Real time quantitative PCR (ABI PRISM 7700) was used to determine the relative amounts of helper and helper-dependent adenoviral vector. Two specific target sequences were selected present either in all helper viruses (Ad5 sequences from 11358-11456) or in all helper-dependent adenoviral vectors based on stkl20 (bp 12037-12176) (Morsy et al, 1998, Proc. Natl. Acad. Sci. USA 95:7866-7871). A plasmid containing both target sequences was constructed and used as one standard for both amplifications. Within each target sequence, a set of forward primer, reverse primer and probe (which is located between the primers and contains a fluorogenic reporter and a quencher) have been selected. Separation of quencher and reporter generated a fluorogenic signal during the logarithmic phase of PCR amplification which was plotted versus cycle number. The plot was used to calculate template concentration based on the software of the 7700 system (ABI).

EXAMPLE 2 Construction Of Helper Viruses

Helper viruses for the helper-dependent system were made based on Ad5. In order to obtain specific helper functions, the plasmid pAdEl-E3+ (containing the entire Ad5 genome with a complete deletion of the El region) was modified in two regions: i) between the left ITR and the promoter of protein IX; and ii) in the E3 region.

To introduce changes at the left end of the genome a shuttle vector "ploxΔpack" was constructed. It is based on pUC (Boehringer Mannheim) and contains the following elements:

A) nt 1-195 of Ad 5 linked to a unique Pac I site immediately before nt 1;

B) a loxP site starting directly after nt 195 of Ad5;

C) a synthetic packaging signal contained in the following sequence: 5'- GTACACAGGA AGTGACTTTT AACGCGCGGT TTGTTACGGA TGTTGTAGTA AATTTGTCTA GGGCCGAGTA AGATTTGACC GTTTACGCGG GGACTTTGAA TAAGAGCGAG TGAAATCTGA

ATAATTTTGT TGTACTCATA GCGCGTAATC TCTAGACG-3' (SEQ.ID.NO:4).

D) a second lox P site;

E) a linker containing cloning sites; and F) 2247 nucleotides of Ad5 sequence starting from nt 3534 (according to wild-type adenovirus numbering).

For comparison, a second shuttle vector, "ploxpack", containing the wild type packaging signal instead of the synthetic one was constructed. It contained: A) nt 1-195 of Ad 5 linked to a unique Pac I site immediately before nt l;

B) a loxP site starting directly after nt 195 of Ad5;

C) Elements AI-AIV of the wild-type packaging signal contained in the following sequence: 5'-GTACA CAGGAAGTGA CAATTTTCGC GCGGTTTTAG

GCGGATGTTGTAGT AAATTT GGGCGTAACC GAGTAAGATT TGGCCATTTT CGCGGGAAAACTGAA TAAGA GGAAGTGAAA TCTGAATAAT TTTGTGTTAC TCATAGCGCGTAATCTC TAGACG-3' (SEQ. ID. NO. 5) D) a second lox P site;

E) a linker containing cloning sites; and

F) 2247 nucleotides of Ad5 sequence starting from nt 3534 (according to wild-type adenovirus numbering).

The second shuttle vector was generated by cloning the 4.9kb Xhol fragment from Ad5 wild-type (Ad5wt) DNA (nt 24797-29791) into pUC. A 2900 bp fragment of human DNA located on chromosome 1 lql 3 starting 13340 bp upstream of STS marker 1 IS 1337 (Genethon) was obtained by PCR and inserted into the Xbal site at nt 28593 (Ad5wt) in either orientation. This fragment contains part of inton 2 of the human LRP5 gene. A strategy based on homologous recombination between plasmids in

E. coli (Chartier, et al., 1996 J. Virol 70: 4805-4810, which is hereby incorporated by reference) was used to introduce those sequence elements into plasmid pAd5El-E3+ containing a complete Ad5 genome with a deletion of El. The procedure allowed for insertion of those elements at exactly the positions described for the shuttle vectors. The flanking Ad5 derived sequences were completely maintained. The following plasmids for new helper viruses were constructed: pAdlspLIl = Helper 14 — has synthetic packaging signal, intron of LRP5 in E3; pAdlpLIl = Helper 1 — has wild type packaging signal, intron of

pAdlpE3 = Helper 11- -has wild type packaging signal, wild type E3.

Plasmids containing the Ad 5 genome with the described modifications were digested with Pad to release the virus genome from the plasmid and transfected into 293 cells by calcium phosphate coprecipitation. Resulting virus plaques were used to reinfect cells. Amplified virus stocks were characterized by restriction and sequence analysis.

1. Growth of helper viruses in 293 and 293cre cells

Different helper viruses as well as a El deficient adenovirus (dl70-3) (Bett et al. Proc. Natl. Acad. Sci. USA 91:8902-8906 which is hereby incorporated by reference) taken as control, were grown in 10 layer NUNC cell factories, purified in CsCl Gradients and dialyzed. The virus concentration (particle concentration) was determined by spectro-photometric analysis (OD260)- L5 x 106 cells (293 or 293 cre) were infected in a 6 cm plate with 100 particles per cell. CPE was apparent in all plates after 48 hours. Cells and medium were harvested, frozen and thawed 3x and the titer was determined in an end point dilution assay. The number of infectious viruses produced per cell was calculated and the results are shown in Table 1.

Table 1

infectious units per cell

The amplification rate for all new helper viruses exceeds that of

AdLC8BHG101uc (Parks et al, 1996, Proc. Natl Acad. Sci. USA 93:13565-13570, which is hereby incorporated by reference) by at least 20 fold. The growth of the helper viruses in contrast to dl70-3 is well controlled in 293cre4 cells. These cells release only 1-2 infectious particles/cell. The rate of suppression is therefore dramatically increased for the new helper viruses Viruses without or with the insert in E3 in either oπentation do not differ much in virus yields, thus, the chosen E3 insert did not have a major impact on virus growth.

2. Amplification of helper-dependent adenoviral vectors over a single passage

A characteπzed stock (known particle titer and helper contamination) of a helper-dependent adenoviral vector containing the human OTC gene was used to infect 293cre cells. Cells were infected with 700 particles of the helper-dependent adenoviral vector and 100 particles of one of the helper viruses per cell After 48 hours cells were collected, virus was released through 3 freeze/thaw steps and puπfied by Cs banding Particle titers were determined by spectrophotometπc analysis (OD260)- Virus DNA was extracted and analyzed by restπction digests (Figure 3).

Overall virus yield was slightly increased for the new helper virus over helper AdLC8BHG101uc. However, the ratio between DNA-contammg particles and empty particles is increased for the new helper. Since helper DNA is undetectable the gel, helper virus does not contπbute significantly to the overall output.

To determine helper contamination more accurately, quantitative PCR based on Taqman™ technology was used. Contamination was similar between the helper AdLC8BHG101uc and the helper LPLI1 containing the oπg al packaging signal, whereas the amplification supported by helper LSPLIl containing the modified packaging signal resulted in a lower contamination (0.29%) (Table 2). This is expected because yields of virus grown in 293 cells are lower than those of an identical virus containing the normal packaging signal. Therefore helper virus which would escape cre-selection should still have a selective disadvantage.

Table 2

3. Construction of modified backbones

Constructions are based on plasmid stkl20 (Morsy et al, 1998, supra) containing a CMV promoter driven gfp (green florescent protein) gene within the stuffer sequences at a unique Swal site (stkl20gfp). In order to incorporate the additional elements into the helper-dependent adenoviral vector, the 3' ITR (130 bp) was extended to 400 bp from the right end of the Ad5 genome. This fragment contains the complete E4 promoter region. However, no adenovirus coding sequences are present. The resulting adenoviral vector is stkl20gfp-E4-promoter.

In a second construct, a 1353 bp fragment from the right end of stkl20gfp containing part of the stuffer sequence and the 3' ITR was replaced by a 3115 bp fragment of Ad5 from a Muni site to the end of the genome (stkl20gfp-E4). This fragment contains the complete functional E4 region.

4. Competition between helper-dependent adenoviral vectors

Genomes for stkl20gfp and stkl20gfp-E4-promoter were released from the respective plasmids and cotransfected with a circular plasmid containing the entire helper genome into 293 cre cells at a 1:1:2 ratio followed by infection with helper virus at moi 1.

The mixture of viruses was amplified up to passage 7 where the viruses were Cs-purified. Five independent experiments were carried out. Virus

DNA was extracted and analyzed for presence of the two virus types. In all experiments a 5-10 fold excess of stkl20gfp-E4-promoter was found (Figure 2a).

This indicates the presence of a s-acting sequence located next to the 3' ITR (right virus end) which supports helper-dependent adenoviral vector growth. An identical experiment was carried out to determine whether the complete element is located within 400 bp from the right virus end or sequences further upstream contribute to this feature. stkl20gfp-E4-promoter and stkl20gfp-E4 were cotransfected with the circular plasmid containing the entire helper genome (Helper 14) into 293 cre cells at a 1:1:2 ratio followed by infection with helper virus 36 hours after transfection at moi 1. After passage 7, viruses from three independent rescue procedures were purified, DNA was extracted, digested with BsrGl and labeled. The discriminating fragments have equal strength for each rescue (Figure 2c). Therefore, the cis-acting sequence supporting helper-dependent adenoviral vector growth is located between nucleotide -400 from the right end and the 3' ITR.

Example 3 Construction Of Helper-Dependent Adenoviral Backbone Containing Alternative Stuffer Sequences The construction of the new helper-dependent adenoviral vectors

(group A) began with the ligation of a 3498 bp from fragment from pSTK120 (which comprised of the bacterial sequences origin of replication and ampicillin resistance gene, the right and left ITRs and the packaging signal) ligated with a multiple cloning site linker (SEQ. ID. NOs. 6 and 7) containing Hindu! sticky ends and cloning sites Eagl, Asd, Bgiπ, Notl, Xbal, Mlul, EcoRl and Kasl. This clone is named "pITRF". DNA from the region encompassed within HUMDXS455A (Genebank Accession # L31948) used as stuffer DNA was amplified from human genomic DNA in four segments of about 4.5kb each using the following pairs of primers: SEQ. ID NOs. 8 and 9 (PCR 1) SEQ. ID NOs. 10 and 11 (PCR2) SEQ. ID NOs. 12 and 13 (PCR3) SEQ. ED NOs. 14 and 15 (PCR4)

PCR1 product was Klenow filled in and then digested with Ascl. It was ligated directly into the Ascl/EcoRV of pITRF to produce "Construct 1". PCR2 product was digested with Notl/Ascl and cloned into the

Notl/Ascl site of Construct 1 to produce "Construct 2". PCR3 product and Construct 2 were digested with Mlul/Notl (double digest) and ligated to produce "Construct 3". The PCR4 product was first cloned into a TA cloning vector. To avoid inefficient direct cloning for very large plasmids homologous recombination in E. coli BJ 5183 was used to insert PCR4 and additional genomic fragments. The technique requires a small shuttle vector containing the region which overlaps with the insertion point in the large plasmid. This shuttle vector was constructed as follows: 2 kb of the junction over PCR3 and vector sequence was amplified using Construct 3 DNA. The PCR fragment was Klenow filled in and then digested with Sapl enzyme and ligated into PABS helper-dependent-3.0, a small plasmid vector (KanR) cut with ΕcoRI followed by a fill in reaction using Klenow and a digestion with Sapl. Homologous recombination between Construct 3 and the shuttle resulted in Construct 4 (C4).

The helper-dependent adenoviral vector part of C4 is 20kb in length and has 3 unique restπction enzyme sites to incorporate 3 different transgenes (Ascl, Notl and Swal).

As a next step, a CMV promoter dπven gfp gene was inserted into the Notl site of C4 by homologous recombination. For this purpose, a shuttle vector was built containing 2 kb of C4 overlapping the Notl site (pNot). The gfp expression unit was inserted by blunt end cloning into Notl and a fragment of this vector was used for homologous recombination with C4. Because a single Notl site is also present in the gfp expression unit, the gene can later be replaced by other genes using this restπction site. The gfp gene is located at a junction between 2 inverted genomic fragments which prevents insertion into the host genome by homologous recombination which would be likely, if the gene was inserted withm a contiguous sequence.

To incorporate additional stuffer sequences and extend the 3' ITR to include the Ε4 promoter, a second shuttle based on pABSHD-3 was constructed containing 2 kb of C4 including the 3' ITR and unique Kasl, Sail, Bglll and HindHI sites for insertion. The E4 promoter was directly cloned as a Hind HI/ Sail fragment into this shuttle vector.

Stuffer DNA fragments AFO, HSU, ER1, and ER2 (Figure 4), were amplified from human genomic DNA by PCR Expand Kit (Boehπnger) and cloned into the Topo-blunt vector (Invitrogene). AFO and HSU were inserted individually as Sail fragments, the ER1 and ER2 fragments were inserted sequentially after Kasl/ Bglll and BglH/ Sal digests. Recombination in E. coli BJ 5183 lead to the final backbones C4HSU (SEQ. ID. NO. 16), C4AFO (SEQ. ID. NO. 17), and C4ER (Figure 4). Construction of the new helper-dependent backbones (group B) was started by replacement of a 8181bp fragment from stkl20 by the gfp expression unit. For this purpose a shuttle vector was built based on pUC 18 containing lkb 5' to the unique Swal site and lkb 3' to the unique Eagl site in stk 120. The gfp expression unit was inserted into the Bam HI site of this shuttle as a Bglll fragment. The replacement was carried out by homologous recombination in E. coli BJ 5183 between STK 120 linearized with Eagl and a fragment of the shuttle vector. This step removes a part of the hprt region from the vector which was most frequently lost during adenoviral vector propagation. This vector "STKSEgfp" is 19.4 kb in length and misses about lOkb of genomic sequence required for propagation as a helper- dependent adenoviral vector.

Fragments AFO and HSU were cloned after Sail digestion into the Sal I site of shuttle vector pSHl-3ITR which contains part of the HUMDXS455A - fragment in STK 120 and the 3' end of Ad5 starting from nt -400 containing the E4 promoter and the 3' ITR. The fragments ERl and ER2 were inserted step by step into the same vector as Sail /BamHl and BamHl/Bglϋ fragments. The 3 resulting different shuttle vectors were recombined with STKSEgfp linearized with Pad to generate HXAFO, HXHSU and HXER (Figure 4).

Example 4

Generation Of Helper-Dependent Adenoviral Vectors Cells from subconfluent plates were washed with PBS, trypsinized and seeded to 6 well plates (Costar) at 3 x 10^ cells per well in 1.5ml medium. Genomes for helper-dependent adenoviral vector were released from the respective plasmids by Pme I digestion and 2μg of DNA were cotransfected with a 2μg circular plasmid containing the entire helper genome per well. A 24 hour transfection was followed by infection with helper virus at 1 moi. Forty-eight hours later cells were lysed by 3 freeze/thaw cycles and the lysate was used to infect 106293cre cells together with Helper virus at MOI 5. Cells were lysed 48 hours later. The same procedure was repeated with increasing the number of cells until passage 6 at which two 15 cm plates are infected. At this stage helper-dependent adenoviral vector is isolated by CsCl banding and analyzed by restriction digestion.

Passage 6 is also used to infect a large scale preparation (1x10 layer Nunc cell factory). Example 5 Competition Between Helper-Dependent Adenoviral Vectors Virus rescue in a competition experiment was performed using the procedures described above and, instead of DNA from a single vector plasmid, a equimolar mixture of 3 vectors was used. Three mixed rescues were carried out: one with HXAFO, HXHSU and HXER; one with C4AFO, C4HSU and C4ER; and the third with HXHSU and C4HSU. No rearrangements were observed in either the individual or the mixed rescues.

As shown in Figure 5 there are only moderate differences between backbones within the first 2 groups with a slight dominance of the C4HSU construct. However, in a competition experiment between HXHSU and C4HSU the latter clearly dominated. Thus, the remaining hprt sequences in HXHSU have an adverse effect on adenoviral vector propagation.

Example 6

Analysis Of Large Scale Preparations Of Helper-Dependent Adenoviral Vectors STK120-EFlamEPO, C4AFO-EFlamEPO and C4HSU-EFlamEPO

To investigate the behavior of the new helper-dependent adenoviral vector in vivo, the gfp gene was replaced in STK120, C4AFO and C4HSU by the in mice immunologically inert mEPO gene. Replacement was carried out using the shuttle pNot by homologous recombination. Viruses were rescued as described above. Large scale amplifications were carried out in Nunc cell factories and the virus was purified as described above.

Since the size of the helper-dependent adenoviral vector and the helper differ only slightly, the purification procedure does not remove helper virus from the preparation. Such a physical separation was not intended to allow for a replacement of gradient based by chromatography based purification in large scale. DNA was extracted from purified virus and the content of helper virus relative to the amount of helper-dependent adenoviral vector was determined by Taqman™ quantitative PCR. We observed a correlation between the helper content and the GC content of the helper-dependent adenoviral vector (see Figure 6). The experiment with Construct C4HSU has the lowest helper content (about 0.18%) compared to a helper content of about 1% for stkl20. Example 7 Analysis Of The Replication Potential Of Different Helper-Dependent Adenoviral Vectors Cells were infected with 100 part/cell of helper and 300 part/cell of the helper-dependent adenoviral vector. Three hours after infection 1/3 of the cells was harvested and washed with PBS. Another portion was harvested at 42 hours and a third fraction at 48 hours. Cellular DNA was extracted as described above and the relative amount of helper and helper-dependent adenoviral vector DNA was determined by Taqman™ PCR (Figure 7).

Figure 7 shows that if the rescue is started with an excess of helper- dependent adenoviral vector, the ratio of helper-dependent/helper is maintained and even slightly increased for C4HSU, whereas it is strongly decreasing for STK 120 and slightly decreasing for C4AFO. This shows that the nature of the backbone has a strong impact on the speed of replication. Whereas the Adenovirus genome coevolved with the adenovirus polymerase, the helper-dependent adenoviral vector sequence was artificially selected. Therefore a less efficient replication is expected. However, the most important determinant seems to be the GC content of the helper- dependent adenoviral vector.

Example 8 Sequence Information The different sequences for the SEQ. ID. NOs. referred to in the application are as follows:

SEP. ID. NO. 1 ATTTGNδGC

SEP. ID. NO.2 ATTTTGTGTT

SEP. ID. NO.3 ATTTTGTTGT SEP. ID. NP. 4

GTACACAGGA AGTGACTTTT AACGCGCGGT TTGTTACGGA TGTTGTAGTA AATTTGTCTA GGGCCGAGTA AGATTTGACC GTTTACGCGG GGACTTTGAA TAAGAGCGAG TGAAATCTGA ATAATTTTGT TGTACTCATA GCGCGTAATC TCTAGACG

SEP. ID. NP. 5

GTACA CAGGAAGTGA CAATTTTCGC GCGGTTTTAG GCGGATGTTGTAGT AAATTT GGGCGTAACC GAGTAAGATT TGGCCATTTT CGCGGG AAAACTGAA TAAGA GGAAGTGAAA TCTGAATAAT TTTGTGTTAC TCATAGCGCGTAATCTC TAGACG

SEP. ID. NP. 6

AGCTCGGCCGATTATTGGCGCGCCAGATCTGCGGCCGCTTCTAGAAACGC GTGAATTCGGCGCCA

SEP. ID. NP. 7

AGCTTGGCGCCGAATTCACGCGTTTCTAGAAGCGGCCGCAGATCTGGCGC GCCAATAATCGGCCG

SEP. ID. NP. 8 ATTGGCGCGCCTTCTTTCTGGGATGATTCAGCATCAACTC

SEP. ID. NP. 9 GATCGTCGGCCGCTTGGGTCATAGACTTCTTTGAGAACCAG

SEP. ID. NP. 10 ATCAGTTAGCGGCCGCACAAGCTAAGATCACAAAGCTGTTT

SEP. ID. NP. 11

TATGGCGCGCCGCTGACACCCAGCCTGGGTGCCGGTG

SEP. ID. NP. 12 TCGACGCGTAGCGCTGTGTGGCCTTGGCAGTTTCCATAG SEP. ID. NP. 13 TCAGTAATGCGGCCGCGGGATCATTCCTGGACTCAGATTGTTCTG

SEP. ID. NP. 14 TATTAAGGCGCCGGGCATGGGAGTGATCTCACCAACTCTGG

SEP. ID. NP. 15 TCGACGCGTATTTAAATGTGCTGGAGTGTTGAGATACTGTAGTGGT

SEP. ID. NP. 16

Helper Dependent vector C4HSU

The sequence is shown without transgenes.

AAACATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATG AGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTG ACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATG TAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACA CAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTT GGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGA GGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTC TAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTT TTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTGATTCGGCC GCTTGGGTCATAGACTTCTTTGAGAACCAGTTATAAGCTATGGTTTCTCTC CACAGAAAAAGCACTTATGGTGTCTCCCCCTTTCCAGCCCACCAACATTTT ACATCTAATTTGGGGGGGTTTTCTGGACCACTTAATACCCATCCATGGATC TCATGTGAAGACTCCCCTGGCTTGAGAAATCACTGTCTTGTTGAAAATGG GAACAAAGCTAAGTCAGATAGCTGGTTCATCAGCAATGACTTTGACCAAG CCTGATCCCACCCTACCCCACCCCACCCCAGTGACCACCCCCCACAATGG AGCACACAACTCTAAACTGGTTTGTAGGTATGTGTGTGTGAACACGCTGA GGAATCTGCAAAACCAAATGGTGAGTGCAAAACCAAACAGTCACGAGTA AATCTCACAACAACCACGTCCTGAGCTGCAGCCCTTGTTGAACTATACCC CACTAGGGCCCCAAGATTTTAGGACTTGTGTGTGGGTGGGACCTCCCCTTT CTATCATGCTTTAGAAGACAGGGATTTCACCAGAATTGAACATATTGAAC ATATGACCCATTTTTTTCAGCCAAAGGCAATTAAAATAACTTCATACTTGA TATCCATGTCAGCAAAAGCTGCAAAACGCAAATGGGTGGCTGCTAAGAGC CCTGGTACCCTGACGAGCACACCAAGTGCTTAGCAACAGTGGTGTCCAAA GGACCAGCTGGAAGCCTGCCTTGATGAGAAGTTGCTCTTCTTTCTACATGA AGGAACACCTCTACTCTCCTGCTTTTAATACCTGAGCTGTGAGTGATCATC TATGTCCATTAGCAAACATCCCAGTGGAGAAGGAAACACTCATACCCGAA ATCTAAGCTACATAGTTGGAATCACTTCAACTTATTGCAATAAACACTTAC TAAGCACCTATTGTGGGCAAGTCTTTGCAATGGATAATAGTTCAGTAGAT ATTTTG ATGT AATATTTGA A AT A AC A AT A A A A ATTGCC ACC ACTG A ATTTA TTGAGCATTTGCTGTGCTTTAGGCACTAACCCAGGTTCTTTAAATATTTGG TCTTATTCGATCTGTATAAATAGCCATCTATGAGAAAGGGACTATTATTGC CCTTATTTTACAAATGAGGCCAATGAGGCCCAGAGAGGTTAACTAAGTTG CCCAAAATCATACAGCCCACTAGTGGCAGAGCAGGATGCAAACCCAGGC TTGCCTCGTTCCC A AGCCC AC ATGTCGTTTGC ATTGGTGTGG AGGTGTGC A TGTGTTTAGTCATTAGCATGTTATATGATAAGCAAGTTTTGAAACATAGAA ACTTAAAATGTGCCATTAAGAAAAGTACAGGCAAGGTTTTCCAAGGGGAG GTGTGGACCTCCGGACAAATTTTTAAGAACTAATTATAAATACTTAAAAA TGGGAATAAGAAGACAACCTAACTACCTGAACAGTTTTAGAGATGACTCA TGCCCACCCTCTAAAACCCAAACAAAAACAACAAAGTCAAGAAAACCCA TGAAATCTTAGCAAGCGATTTCTATGTACTTGTGAAAAGGATTTCTTTACC ATTCTAATGGGATTTATGCCAACCATAGAGGGCTCAGTGCCCCTCCCATG GGGTGGTTAGTGAGTACAGAGCTGAGCTCACCGGCCATCTGCAGCTTCAT GTTATCAAGCTCCAGTTTGTCCTTGGAGCAAGGTTATCTGGGACATGAGC AGAGGCATTGCTTTCTGCAATGGACAGTTCTTTCTGCCTGCATACCTAGCT CCTTGATAACTTTAAATACCATTTTATAGCCACACTGGAGTTTTGAAGACC TCAATATGCAAATATTACTCAGGTTCTGATTACTTGTCTGCTCCATGATAA CACACTCTAAAAGCAATGAATGGGGCTTATTTGTAGAGAACTGAAGCATT TTAAGCTTTTGCTCAGGAATCCCTGGTAGCTTCCTGTGACTTGCAAGATAT TAGTGATGGGTCAAGAAACAGGACCCCCCATCAGCATAACATACGCAGTG CCTCAGTAGTCCATCAGGCAGAAAAAACTGCAGATGGCACATGGAAATG ACCAGCGGCGGAAGATACCCCGACAGTGTGGGCAGTTCTATTTCAGCAGC AATCAAGAGGGGGCCTGGAGCCACTCAATCAAGTGGAGCAGGATGGGAG CAAGCACTGTGCAGACCAATGCAATCCCCAGTTAACACAAAAAATAAATA AAAGAGATGAGATTCAGTCTCTTGACTGTGACTGACTGGGAGCTTTATAG CTGATGCTTGTGTCTTTTCTCCATTTTATTTAATTAGGAAAAGAAATGCTT ATCACACACTCTACGTGTGAGGTACAACCTCCACAGGAAAGGTTGTTAGG AACATTTCAACTTCTAGAAGTTTCTAAACATAAGGTAAATCCATCTTTGTC CTTGGGATCACTGCACATCTCAGAAAGGCAAATAAATCAGTAATTGGTGG GC ATA ATTACTAGCTCATGG ACTGAC AAGGTCTAC ACTATTTCGAATCTCA CAGAAGTAAGCCATGGGACAGATAGAGTCTGATAGTGGTGCCCCGTTTCC TGGAGGTCACACTTACTCATCCCCCTGGACCCTGGGCTTCTCATGATTGTC AGAGAGTTTGCTGGAACCAGGTCAGCCCAGTTTCCCTTCCCCTGAAAAAT CCTCCAATGGCTCGCACCAAGACTAGAGATGCAAGTGCAAGCACATCCAC CCTCTC AGC AGCC AGGTTTTGCGTTCC AT A ATGTC ACGTACCCCC AGTC AT ACCAATCTCCTTGGAGCTCTCCAGACAGGCTGCCATGTGTGGTCGGCCCTC TGTGCTTGTGCTCCTTGGTTTGCCAAGCCTGGAATGCCCTTTCTCCATGAT TGTACTCTGGGAATTCTGTGTGTCTTTCGAGATGAAGCTCCTCCACCCTGG AAATTCTGCCTCTCTTTCCAGGTCCAGCTCCTGTGGCTGAATACCCTTGGC TGACTGAACATTCTTGCTGGGCCAAGTCTTAATCATCTCTAAATCCCTCTG GTGCCCCGACAGTATCTAGCCCAGGCAAAGGGCTCAGCAAATACTTGCAA AATTCAATAAACTTTACTATGTTACTAGCTTGGCATGATGTTCTAGGCACT TGGGAGATTATAATCTGGTGAGCATTGTGCTAGTCTTTTTTTTGGGATAAA CACCAATGATAAACTGAGCCTTTACTCTCATTTTAGCACATCTACTCATCT CTTTTCAATGCTGGTGGGTTTATAAAACCCATCAGTAGAACAGAGAGTGA TCATAATCATATGGTGAAAATAACATCAGCTAACATATTTACTGAACATG CTTTAGTGTGCTGGGCACTGAACTCTGTGCACATGTGAATTTGAGATAGAT TGTTCCTAGCTAATAAGATGAGGAAGTGGAGATGGGCTTACTCAAGTCAC ACAATAGCAAGATGGGGTACAGACAGAAACCAAGCTAGAAACCAAGCAA CCCCTGGGTTTGGAAATGCATGGGCTCCTCCTCTGCACATGGCGAGGAGC AGTCAGGTGCTCCTCCTTCTCTCATACTGAAATAACTCTGCACTTTTGGCT ATTCTGTGACACTCTGTGCTATTCTGTAGCTCAAATGGCTCTGGTGCAAGG AGCTCAGATTATGACTAACCCCCATAGATATTCAGCTGCTTTGCAAGAAG TGGATGAATGCTCTGGCTTTATAAATTATTGACTAGATTGGATATTGGCCC AATCTCCACTCTGGTGATTCGGAAGGAGGCATATGCACATTTGCAAGGTT ATAGTAGTGCACTCTAATTCCACTGGCTTCTGAGAGCTTGTAGGTTTCTTG ACTTAATCATTCTGGAATTAAGGTAATGGATCCTCAACACCTTTTCTTTCC CTGGCCTTTACTAACCATGTAACAGAAATGAGCAGAGAAAACCCAAGAA AGCGAACTGGAGACTTGATGAGTGTGTCAAAGATGCTCAGAGTCCAAGGT CCTCTGTGGCTC ACTG ACTTC AG A AGCC A ACCTCCGTTGTTC A AGTC ACTT GTGAGGTTACTATGCTAGAGCACTAAATATTATCCAGATAGCCCAAAGAG GTGAAGGCAGACATGTGGAAGAACCTGGATTTTTGGGCAAGTTGATGAAT GCCTCCTGCCCCATATCAAAGAGAGGTGATAGGAGACAACTTTGTGAATT TGAAATAATGCACCGGGGAAACAGGAAAACGTAATGTAAGTCACGCTTCT TGGCTTTTTTCTTGCC ATTACCCTACTTGGCC A AGTGC A A ATGGGATTTC A ATATATCATAAGTATGCATCTATTAATAACAATGCAAGAAAGCTGTACAA CTGAAGTCTGAGATTTTGTAAGAAACAGAATCTCTGTAAGCATCACCATC CAACAGAACTTCCTGAGTTGATGCTGAATCATCCCAGAAAGAAGGCGCGC CAGCTCTGCAGGATCTTCAAGTCTGGGGTGCCACCAGCAAGCGACGGTCC TCCATGGGCTCTTCACCTTACGGCAGTGTCCAGAGGCACCGCCAGTCCTCT GCTCCTATGCTGGTCCTGCTGTCCCTGGCAAAAGGAGCCAGAGCATTCTCT CCAGGCCTCCCGAGGAGGCTGCTTCCTTTGTTTTGCAGATGGAGGCTCCCA TCCTTTGTTCTGAATCAATGTGCTCCAAAGATAAGCCCCAAGAAAACAGT TGTTGCCTTTTGACACTGACAATTAGAATCGTTGGAAAATGGAGAAAACA GGAAATGGCAAATGGTTTCAGTGACCAGGAGGAAACCGTGCCTGAAAGTT GCTGCTTAGTGACTGGGACACTCGCTTTCTGCTCTCTTATGAAGGACAGCC TAGGCCGTGTGGCCTTTTATAAACAAAGCTATGAAGGGGTCGTCAAATTT TCTAGGGCTGCAACTGTGGCACTACGTCCTGTTGTGCCAGGTGACACTGA CAAGCAGCACTGAGTTCTATGCAAGCCCAGGTGTGCTTCTCTCATGGTGA CCCCCAGAGAACTAAGGCCCAGCTCTTCCTCTGTCACACCCCTCCCAGCC CCCACTGTCAGACAAGGGACCACATTCACAGACAGTCTCAGCCAAGATGG CAACCTTGGAAGTCCTGGGGATGCCTTTCTAGAAGCTTTAGGCTGACGCC AGGGAGCTGTAAAGCCCCCACCTGTCCCTGGGGGTTGTGTGCTGGGCAGG AGAGAGGGGAAAGAGCCCAAACTCCACCACTGCCTGCTGGCACAACTGA GCCCACGCCAGGCACTGCACCAGCTTCACTCACCAGCTACCACATGCTAA TGTCTCCTGTTTACCCAGGTGCAATCACCTGAGTGCTGCTTTCTCACCACT GCATGGGAAGAACGATCTCATTACCAATTCAACCACTTAAGCAGGCAAAA GGACTCCAACGGGGGAAAGGCAGAGGACAAACGATTCGGGGACTGAGAC AGATGCCCACAAAATGTTCCTGGATTTTAGCCGGATTCGGGCTGAAGTTC CTTGCCCTATGAGCCCTTGGAGGAGAGCATCCTGACTCAGACAAGAATTC AAATGACAAGACTTCTGGGGGATGGCAGTGAATGGATCCAAAGCCTTGTT TTATAAAGAATGTGGAGGAAGGGAGGAAGGAAGGGAGGGAAGGGGAGA CTGAGAGGGAGAGAGAATAAAGAAGAGGAAGAGAAGGAAGAGGAGGAG GAGGAAGGGGAGGGGAGAGGAAAAAGGGGAGGGTAGAGGAGGAGGGAG AAGGGGAAGGGAGGACAGAAGAGGGGAGGGAGCGGGGAGATTATCTAC CCCTCCATGCTAGGGAACTCCTGTCTCCTTTTCCAGCAGACACTGCTCCAT GAGTGCCCCCACAGAGAGCCAGCCTGCCACACAGCAGAAGGTGCTCAGC TATCTGTCCCAAAGGTTGAGCACTAGTGTATACCACAAGGAAAAAAACCT TCTTTTGTACTGTTTGGAAGCCAGAATTAGAAACAACTCGTTCACCCTTCT GACTTTCCCTTTGCAATGACAGGAAGATAGTATACTGCATGGGACCACCA CACATTCAAGATCTGAGTTGTGGGGACCCCCACCCCCACATGGGTGGAGC CCCCTGAGACCCGTGGGCTCTGACTGGCCATGGCCCATAGATAGTGACAA CATACACACTGCTATCTGAAGCCTAGTTGGAGGACGGATGGATGCTTTTA GGAAAAAATTCCCTTTAGTCGTCCCTCAGAAACTACAACGGCTTCCTAAA ATGAATGGCAGGCAAGTTTTGATTGTTTCCTAAGTTCCCCCTAACTCTCAG AGCCTGTGACTCAGCTTCTCCAGAATGCCTGCAGGGAGGCAGGACCACTG GGAGTAATGAGCACGAATTGGGCCACTTCTCCCAGTGGACTGTTAACGCC GAGATCTGGGGTGGAACCCACTGAACACCTGCCCTTTTGCGTTGCCCAGA ATGCAGCCAGAGGGATACGGGCCACAGTAACTCTGGGGCCCCTGAACCTA CCTTCCCAAGACCCTGACGGGAAGGTATTGCTTACATGACAGTCCAGTCA GTCCCTCTGTCACTCAGTTAACAAACATGGTCATGGGTCTACTATGGGCCA GGCTCTGTTCCGGGCATGGGGATAACACCGTGTGTCTCTACTCTTATGGAC ACAAGTAAATCAATCAAAGCAGATAATGTTCTAATGACCACCCAGTAGGG TGAAGGGATGGAGGGTGGGCAGGTGCTGGCAGAAACCTAAGGTCAAGTC ATGGGACCCCAATGGGGAACTGGAAACTGCCACACCATTTTCTATGTTAG GGGTCTTTCCTTGCCCTTCTAGAGACCTCACCCATGGTAATGATGCTGCCC TCAGGTTTGAACTGGTCTGAAGCTGTCACTGGGGGCTTCACAAAATGAAT GCCACTGAAAGCAAACGGCAGAGATCTGTATATGAAATTCTTTTTACCCT TTGTTTTCTGTCTTCCCACATGATCTATGACTGCTGCAAACTCTATTCTGAC TCCTCCTC AGTCTTGGCCGGTCTGGG AAGAACTTGGTTT AG ATGCTGGGA ATGATCCCGGGCAAAGGAAACAAATATATTTCCAGCTTGCTTTTCTTCTTC CCCTGTGGTGCCTGCATCCTCTGTCTCGCACTGACTCACAGGCTGGACTGA CAAAGTGGCTGTGTTGACAGGGAGGATTTAGTGGATGCCAGGCCTGACCC TCAAAGGCCCTCAGGTGGGGCCAATTTCCAAGGTTCTTAGAGAAGATTTG GGGCC AC AAATCTGCTCTGCC ACTTC ACTGCTGGGTGAAAGCTGTTGGCTT GAGAAGAGAGAGAATAGGTGAGGGACAATAATTCTCAAGGCAAGCTAGC TGATATCCTTTCATTTGTGGCTTCCAAAGACAGGGAAAGGCAAGGCAGAC CAGGGTGGAAACAGCGACATCCTGAGGGGACGACGTCCGTTTTGCTAATT CTGTTTCCCTGGCAGCTTTGTGCTGGACAAGATTTCTCTGGTTTCAGCTGA AGAGTGAACAGCTGAGGAGCTCTGTGACTCTGAGAGAGAGAGAAATCAA TAGCCCTTGAAAGGAAAGCACTGCTTGTTCTTCCAGGCCAGGGCAGGCAG GATAAAGAGAACAAGGCTGTACATACCAAAGCCGAATTTCCATTAAAGG CCAGAATCCTGGGAGGCCCTTTCAGCTTAATGACCATCTTCTGATCACTAT CACTTAGAAAATGTTCATGTCTCCAGTTTTCTCTGCAGATAGTGCAGACAT CCTGTGCACATTGATATTTTATGCACCAGATGCAATTCTCTGCAAAGGTCC CCTCTGGCTGGTGTCCATCCGCTTGCTCTCATGACCACTGATGCGACTCTG GGGGCAAGTCAAGCCATTCCTTCTAGACTCTAGAATCTAGAGGATGATCC CAACTCACAGGCCTCTTGGACAAGCTTGCTTTGGATTCCTTCTGCCTGTAC TACCCAGACTGCCAGTCCTCCAGCAGGGCAATCCAACTTCCTGCCTCTAG GGTGTGGGAGTCCTGGGCGGGGTTTGGAATCCACAGCAGATGGCTGGCAG GAGAGGCAGTTCCCGAGCACCCACTAGGTGCCAAGCAGGCAGGTATCAG GTGCAGGGCTTCGGAGGTGCAGAAGCACAGCCCCTGTCCCCCTCAGGTTC CCAGTCTGGCCTCAGGGACAGACAGGCAGGTAGGTATGGAAACGGGGTA CTGGAAGGCATACAGCTGAGGGAGAGAGCAGCTGCACCCTGGTCAATCC CCCACTCATTTCCCTGGAGGTGGGGCTGAGGCACCTGGAGCACTCTGCTT GCTTGGGTTGTCCCTTTGCCAAAGAATGCCGGGCAGAAAGTAGAGAACAA GCAGGCCCAGGAAGCACTAGAAGTCCCTCATGGGTGGACAGAGCTTTACC TTCGGAGCTGTCACCTCCTGTGTTCCTCCCAACAACCCTGTGAGGCAGGCG GGCAGGCTGAGCAGGGGACTGTGCCCCGTTGACAGATGAGGAAAGCGAG GCTCAGGGGGAAGTGACTTACCTGTGGCGTCGCAGCTAGGCAGTAGTGGA GCGGGCACCTGCCTCCAGGCCTTCGGATCCCAGCTGCCCCAGGGCGCTGC CTCTGAGGTGCTGTACGAGGAGGTGACTCGGGCCAGCTCATGAGCAAAGG AGCTGCCGGGCAGGAAGGACTCACTCCCCAGGGCCTGGCAGGACGGGGG CTGTCGCCCGAGGCTGACCTGGGGGTGCCTGGCTTCCCCAAGCTTGCAGG CTTTGTCTAC ATGAGTCTACAAC AACCGATTGAAC AATCC ATC AGCTGCTT GGCCACAAAATGGTTCTGACTGATCTTGTCACTGCCCTGAGTGTCTAGCTG GGTCATCTGGGTGTTGTGGTTATACCCACCAACTGCTTTGGTGGAACGAA ACCGCGGCCGCGGGATCATTCCTGGACTCAGATTGTTCTGAAGAAGCCCA GTTCTGGGTGGCATCAAGTGCTTGCTAGATGGGGGGCTTGCCTTGATCCG GCTACACTTGGAGGTGACTTGTTCTTGGACGGCTACATACAGAAAGAGAG AAGTGGGGATGAGTTCCAAAGGCATCCTCGACTTCGGCTGTGGCCACCGG AGGGTAGCTCCTGGCCCAACACGGACTTCTCACCTCCCGCCCTTGGCTCTC TACTGAGCTCCCCCCTGCTCCCCAATTCCTCGCCATTCCCCTCATTTCTCTG CCCTCAGCCTGGACTGCAGTTCTTCTGGGAAGCTGCCCCAACTCCCTAGGT CTGTGCTCACCAAGAGCAGATCACACTGGACTGAAATGCCAGCTGATTTG TCTCTTCAAGAAAATTGGAAGCTCCTGGAGGTCAGGGTCCATGTCTGCTTT TACACTCAGTGCTCTGTATGCAGGCCTGGCACTGCCCACCCTTTGACAGGT GGTGCATATTTTGTAGAAGGAAGGAAGGGGCCAGGTGGGGTGGGCTGGG CTGGTGGCGGGAGCTAGCTCAGCCTCTTAGATTCTCTACCCGATGGATGT GACCTGGGACAGCAAGTGAGTGTGGTGAGTGAGTGCAGACGGTGCTTTGT TCCCCTCTTGTCTCATAGCCTAGATGGCCTCTGAGCCCAGATCTGGGGCTC AGACAACATTTGTTCAACTGAACGGTAATGGGTTTCCTTTCTGAAGGCTG AAATCTGGGAGCTGACATTCTGGACTCCCTGAGTTCTGAAGAGCCTGGGG ATGGAGAGACACGGAGCAGAAGATGGAAGGTAGAGTCCCAGGTGCCTAA GATGGGGAATACATCTCCCCTCATTGTCATGAGAGTCCACTCTAGCTGAT ATCTACTGTGGCCAATATCTACCGGTACTTTTTTGGGGTGGACACTGAGTC ATGCAGCAGTCTTATGGTTTACCCAAGGTCAGGTAGGGGAGACAGTGCAG TCAGAGCACAAGCCCAGTGTGTCTGACCCACCCAAGAATCCATGCTCGTA TCTACAAAAATGATTTTTTCTCTTGTAATGGTGCCTAGGTTCTTTTATTATC ATGGC ATGTGT ATGTTTTTC A ACTAGGTT AC A ATCTGGCCTTATA AGGTTA ACCTCCTGGAGGCCACCAGCCTTCCTGAAACTTGTCTGTGCTGTCCCTGCA ACTGGAGTGTGCCTGATGTGGCACTCCAGCCTGGACAAGTGGGACACAGA CTCCGCTGTTATCAGGCCCAAAGATGTCTTCCATAAGACCAGAAGAGCAA TGGTGTAGAGGTGTCATGGGCTACAATAAAGATGCTGACCTCCTGTCTGA GGGC A AGC AGCCTCTTCTGGCCCTC AGAC A AATGCTGAGTGTTCCC A AGA CTACCCTCGGCCTGGTCCAATCTCATCCCACTGGTGCGTAAGGGTTGCTGA ACTCATGACTTCTTGGCTAGCCTGCAACCTCCACGGAGTGGGAACTACAT CAGGCATTTTGCTAACTGCTGTATCCTAGGCCAATAAATGTTGATCACATT TATAGCTGCCATGGTAGGGTGGGGACCCCTGCTATCTATCTGTGGAGGCT CTGGGAGCCCCTGACACAAACTTTCTGAAGCAGAGCCTCCCCAACCCCTT TTCCATTCCCTATACCTGACAGATGGCCCAGGAACCCATTAGAAATGGAA GGTCACTGCAGCAGTATGTGAATGTGCGTGTGGGAGAAGGGCAGGATCA GAGCCCTGGGGGTGTGGCAGCCCCCAAGTGATTCTAATCCAGATCCTAGG GTTGTTTCCCTGTCCCATTGAAATAGCTGCTTTAAGGGGCCTGACTCAGGG AAATCAGTCTCTTGAATTAAGTGGTGATTTTGGAGTCATTTAGACCAGGCC TTCAATTGGGATCCTGCTCTTAGAGTTGGATGAATTATTTAACTGATTTTC AGATCTCCTCTTTCTCAATGCTTTCAGAAGCACAGTAACTGCTTACTCTGA AATGAATTCTCACCCCACTTCCACATATGCACCCCTTGCCCACCCCTTTGG GAACACTGGCCTTAACTGCTTACCTTCAAATGGACTCATCTGTTGGGAGAT ATATGCATTCTGCCGTTCAGGGGTCATTGCCATAAGACCTGATCTCTGTTC CTCTTGCTAAACAGAAGATGAAAAAGACAAATTAGATTACAGCTACCAAT TAATAATTAGCCTTAGGATCGCTGCGTGGGGACCTAGGACTTGGCTTTGG TGCAGCAGAAAGCATGAATAAACACACCAGCATACACTCGCATGCATGCC CCACCCTCTCGAGCAAAATTCCACAGGTATAAATAAAGTAAGATTCTGCA CCTGGGTTAAAAACACAACTGCAACAGCATAGAATGGGGCAGGAGAGAC AGAACTTAATAGCAAGAGCACACAGAAAAAAGTTTTAGGCATTTTGGATG TCCATCTGCTCAGGATGGGTCAGCAGTGAGATGCGGTCACCAAAAGAACA AATGTAACATTAGGCTGCATTAATAGAAGCAGAGTATGTAGAAGGAGGG AGGTGACAGTCCTATGCTAACTCTGCCTTGGCCAGACTATACCCACAGGA GTCTGGGC ATGCCAGTCTC AGGGAGACCC AGAC AGACTGGCTGC ATTCAG AGGATGGTAAGTAATGAGAGTGGGGATTGGACTTCAAACTACCCAGACA AAGAATGGCTGAGCAAGCCAAGGATGCTGTGGCTGGGGCAGAGCAGACT GTGGGCTATGTAGTGGTGGATACCTAGCCTCTGCAGGGCTGTCATAGGGA AAGGACATTGAGAAGAGGACTGAGGCTTGTTCCTGGTGGTCCTGGCATGA ACGGCC AG ATG ATC AC ATGGTC AGGTGG AC AC AGTCTCC A AC ACTGGG AG TAGCCAAACACTTACTGCCAACCTCCCGCCCTTCTCCTGACTAGTTGCAGC ATAGGCAATTGGGAGGAGCTTCCTGTCTCCATCTGAAAGCTGGCTGGGTG GGCAGGGGGAGGAGCGAGCCAAGTTTCAAGGCCGCAGTTTCAGCACTCA GTCTGGGATCGGCTCAAGGAGCAAAGGGGAAGAACATAGCCAGGAGGGA ATAACATGAAGGCCCCCAGACCCAGAAAAGGCATGACTTGCTCTGAGACC CTCAGCCGGTTGGTGTCAGGTTGTGACTCGGATCCAGGTCTGACTCCCAGT CCAGTGCTTGAAGCCTCACCCCACACAGTGAGGGGAGCCCGGCCATCTCT GCTCAACTGCTGCCATCTCTCTCCCCTTCTCAACCACCAAGGCAGCTCTGT CTGGGAGCACAAGCTCCAAGTCCACTTTCTGGTCTGTGTCCCCCCCAAGAT GCCAGAGGACTTGCCTCTACAACACGGGCTGCCCGTGCAGTGCCTGCTTT TCCAGCAAAGGGCTTCTGGGAACCCTTCTCTGCACTCAGTGGGGCTGGTG GGAGTGGGGCGGGGTAGCGACCCAGTGCTTGGGACTGTGCCCAGCTCTCA GGCCTGGCAGCAGTTCCTGGCCTTGGTTCCTGCCAAGGCAGAGAGGACAA ACACATGGCACCGGGAAGACTACACCAGAAGCGATTCCACCAGACTGGG GTTTGCTTTTCTATCCCGCCCTTAGCCTGCTTCCTGTCCTGGTCCCTGCCTC CCCCTCCACTGGAGCTGCCGTGTGGGCAGTGAGGGGCTGTTTCTCAGCTG CCCTATGGAGCTGCCCTCTCCCTGCCAAAGCATTGGCAAGGCGGCAAGGG GTGGGGGTGGGGATGGGGGGTGGGATCTGCCTTCTCAAGCTCTCATTATA CTGAGCACGTCTCACCCATTATTTTATGTCATCTAGCAACACCCCATGTGG ACACTGAGGAGCATGGGGGTCACATGACCACTGCCCAAGGCCACACCATC CGGATCTGCCTGAGATGGTCAGGGTTGGCAGCCATTTCTGAAGGCAGTCC TTTCGCTTTGGCTCTTCTTGTACCAGTCTCAGGACATCAGGGCAGAAGATC TACAGTCCCCAGCTTACTGATGTGACAGCAGAGGCTCAGAGAGGTTAAAT GACTTGCCCAAGGTGACACGGCTAAGAAGTACAGTATCTCCTAACTGCAG ACCAGGTGCTTCTGCTGCTTCTGGGGACAGATTCCTGCGTGGCTGGCTAG GTCTAAACGGTCCTTAACTCCATCCCCACCGGTTGCTGCATTAGTTTCATC AAATAACACAGTTGTACAGAGGTAGGGGTTCAGGGGCAGGGGCAGATGG AGGCTGGAGAGTGTGACTAAGGAAACAGCAGGGGAAGTGCGGTAAAGTC CGAAGGGAGGGACGGAAAGAGAAAGCCAAGCCCAGGGGCGTGCCAGAC AA AAGG AA AGGCC ACGCCGGGGC AGGGC AGGCTTC AGCGGGTGCTGGGG CGTCTTCATCCCGGGAAGCACACATTCCAGAGGACCCCGGAGTCTAATGG AAAAGCTGGCCAGCCTATCACTATGGAAACTGCCAAGGCCACACAGCGCT ACGCGTATTTAAATGTGCTGGAGTGTTGAGATACTGTAGTGGTGGGATGC TTAAGTGCTGGGGTGCTGGGTGTTGGGATGCCTAGGTGCTGGGGTGCCGA GATGCTGGAGTACTAGTGTGCTGGGATGCTGAAGTGCTGGGGTCCTGAGA TGCTGCAGTGCTAGGGTGCTGAGATTCTGGGCTGCTGGAGTGTTGGGGTG CTGGGATGCTGGAGTGCTGAGATGCTTGGACAATGGGGTGCTGGAATACT ATGGTGCTGGGGTGCTGGGGTATTGAGATGCTAGGGTACTGGGATGCTGA AGTGCTGAGATCCTGGAGTGCTGGGCTGCTGGGCCACAGGCTCTTGAATC CATTCGTCTGCCCAGGGGAAGAAACCAGAAGATAAAGAGCTAATGAAGG AGCTTTGGTTGAGAGGGAGGAAGTAATGGAAGGAGCAACATCTTGTGGA GGAGCAGGAGAGAATGGACCTCAGGTTGGGAGAGAGGGCCAGGCTACAG GCCAGAGAGGCAGAAGGATTCCAGCAGAGTGTGGGCTCCAGGAGCCAAG GGGAAACAGGTTTCTGGGAGGAGAGAGTCCAGTACTGCTGAAGTGGCAA GTCCGCTGAGGACCAGGAAGCTTCATTTGGCTTTATGACCAGGAGGAATT TGGAACTGTGACTAGAGTACTTAGGGGGAAGGAGGCAAGACTGGAGCCA GATTGCTCTGGGTTGAGGGGTGAGTGGGAGGTGAAGCAGGGCACTGTCAC TCCTTTGAAGGGTGGCAGAGAGCTGGAATTGGTGCTGGATGGGCTGTGGG GTGACAGGGTCATGTGGAAAGCCCCTGGGGGGCACCTGGAAAAGGAGAA GCTGACAGTACAGTGAGAGGACAGCTAAGGGAAAGCGGAATGGCAGAAC ACGCACTGCCAGGAGGAATGAGGATAGGGTCAGGAGTGCCAGGGGCAGT GAGGCCAGCCTGGGGTCAGGTGGCAGGACGTGTCCAGGAAGCTGGTCTG CACTGCAGCCCACACTGGCTCAGCCTTGAGGTTCCCTGTGTGGTTGGGGT AGGAAGTTGAACCCTCTGGGAATGGAAGATGGAACCAGCTCTGCGAGCC AAGCTCAGCTTTTATCTATGGGTCTCTGAGGGCTGGCAGAGCTGAGTGGG GACAACTGTGATCCGTGAGGCTCTCAGGTTGAGGTGGCCCCTCCGGGAGG GCTTCATTTTCCCAGCGGGTAGGTTCTAAGCAGCAGTGGCTGGGCAGGTG GGTCCAACACAGAGCCAGAGAAGGGTGAATGGGCCTCCTGGCACCCCAC CCCTGCTGCCCCTGAGCTCAGTGATGGAGGGGGACAGCACAGCTGAGCCC AAGTGCTTTGGTGTGGCCCTGAGGGAAAGCTGC AGCCTGCCTGGGGCCTG GCATGGATGGGACACTTGAGGCAGAGGGACAATAGTGGGCGCTGCAGTG AGGCTGGCTCTTGGAGAGGTTTCCTGAGGAGTGCTGCCTGAGACGGGCAG GGAGAACAGAGACAAAGTTGGTGACAGGGAATGAAAGCTGACTGAAGGA CTTTACCCAGACCTATGAGGATATCTCTCTCAGCAGGAAGCAGGAGGGGA CTGTGTGAGGACTGGCCAAGAGCTGGAGTGTTGGGAAAATGACTCTTTCT CCGACCCCTCTGTCCTAGCTCTGGCCCCTGGACTGCGGAGGTCTGCTTCCA CCCCCATTGGTCGATCGTTGTCCCTTGTCACAGCCATTGAGAATTTTGGCA GGGAGCATGTTCTTAGAGCATTTTTAGGCTCTGCGGGACATAACAGCTCT GCCTCAGAGCACATGCCTTTCTCAGCTCCTGAAAGCCACTGATCAAATTG GAACATTTTGTACCTTAGGGATGAGGATATCAACTCTCCCAGCCACTTAG AGGGATAAATGTGATGATGCATTCAATTGTGACTACATCTGATCCCAACT GTTGCTTCAGCTGCTCTCCTATAGCACATGGCGGGAGGCGTGCATCCCAG TAGCTACCTCCCCACTTTTGGGGAGATGTGGTTCCATCCATGAAACCTGGG TACCCGCCTACCAGGTCCTGGCCTATCAGGTGGCAGGGTCTGGTCAAAGA AGGGCATGTGTGGTCTTCAGCAAGGGAGACAGGACGGTGGTGCAGAGCG TCTAGACCCTCAGGGCAAGTCTCCCCCACACCTGCTCCCGGGGCAGTTGT CTTTGTGACCTCCCATCCCCCTCTGTTTCATCCTCTATAAAATGAGGGGCT GAGCCCCAAAATAACAGGCTTCTTTGCCATGATGCAAAACTGCTGAATCT TTCTTTCTGACACACAAGGCATCGAGCAGCCTCTGAAAGAACCAAAGCCA CTAGCAGGCTTCCTGACTTGGGTTTGTAGGTACTGAATACTCCCTTGAAAA ATAAAAACATAGAGGCACTTTTCTCCTGGCTGTTTATTACAGAACGAAGA AAAAACACACTGGCTTGAAACAGACGCCAGATTTCAAATGTAGAGGTGA AATACGAGGTGGCAATTAAAATGTGATTACAGAAAGTCTGGACACTGAGA AAAGTTTACAGGACAGTGGGTGTGGGTTTTCTATAACAGACACTTAAATA TACATGACGATAATTGCAGATAGAAACCATCAAAGACAAACCCCAAATC AACTAATAATGTTTACAGATGTTCCCCCCCAAACCACAGAGCCTTACATC AAAACAAATACTGAAAGGCTTTAAACCAGGAACAGCTCGCCTTAACCCCA CGAGGGTGCACACAAGCTGGGCTTTTTCTCTCGGTCTGAATGGTAAAGGG AGGAGGATACTCTAGCTCCTCCAGGTGGATTGCTGAGACAGGGCTCGGCT C AC AC ACTGTCTCTGCGCCTCTCCC AAATCTGG AG AACTCTCCC AGCCTCC TGGTAAAGTGTCTCTGTGGGGCACTTAACGATAAAACAGCTTCTGCTGTA AAGCTCATTAGGAAAGAGCTAGCGGAGACTGAAAGGTTCGCAAAAGAGA TTAAGAATCACACAAGGCAATAGGATTTTTAGTGAACATAGAAATAAATG GCCAAGTGGTTTTCTATTTGGCATTTGTCAACTTGCACAACAACTCTTGGT CATATCCACATTGCTCATTGCATTAAAACCATAAGCGACTCAGCCACCTA GCTTAACAAGGTATCACTGGAGCAAACAACACGGTCTGCATATTTGTAAC ATTGTATAATAAACACAAAACAATGCATAGTAAACACAACTCTACTGAAA CAAAAGCCGTCGCTTTATTTACAAAGTCACAAAATGAAGTATAAATACTT CTGTCATTAATGTTTAGGAAAACCATTTACAAAATTTTCAAATATGTACAC GTAGCTTGAAAAATCACCAGCTTTCCATTTTGTCACAGGTAGAGAGAGGG ATAAGCATGGGCTGACAACACCACTCAAATTGTAACGGGAGACAACTGC GGGTATGGATCGACACCACTTCCTAGAGTGATGTCACCATGGGGGTTTCT ATGGGCATCCTGCTCAGATTTAAAGTGCCCCAGCATCCTGGGTGACTTGC CCAGAATTCTGGGCTGTGGCATTTTGAGCAGCAGCATGCTGTTCCAAAAT GTCGTCGATCAGCCTCAAGTTGCACACCCAGTCTTCATCTGGGCTCACACA GGAGCCTTTCAAGAGAGCTTCAATGAAATCTACCTCATTGCAGTCAGGTG ACGAAATCAGATCATTTAGTGGGGGTTGGGGCTGGCGCAAAAAGTCGGCA GGTGGCAGCTCAGGGGGAATATCCGTTCTGTCGAACGGACCTGGGAACTG GCTGGCAGCAACGGCAGAAGCAGCAGCAGCGGTGGCAGCAGCAGCCACA TAGCTTGGTGGCTCGATGCCCTGTATGGGGCTCAGGGGACTAAAGCTGGC CATACCCTGCTGGAGGAACTTGGTGGTGTTTGCTACAGGCACCGGGCCCT GTACCGGGCTCTGCCTGAGGCTCTGGCTGCCCAGCAGGCTGAAGCTGGGG TTGTTGGCCAGGGGCACTTGTGTTCCCATCGCAGCGGGCACTTGTGCCTCC CAATCAGATGGCCTCTGAAGGCAGGCCTGGCCAGAAGGTGAGTGCTGCTG A ACGCT ATTATCC ACTTGGCTGAGGGGTGTTTTCCCCGA A ACTGCTGTGGT CACAGCTGCTGCCGCTGTGACCCATGCAGCATTGTTGAACGCAGTGGGCA TTCTTGGCACACTAGGCCGTCTGAGCTGGTGGGGACTCAAGGACTGGGTG CCCAGGGAGCTGGGACAGAACCCAGGCAGGGGCACTTCTGGTGGGGTGG CCTTGGGGCTCTGCATATGCTGGCAGACAGAGTCAAGTCTGCCCAGGGGA GTCTGGCCTGAGTGTGAGAGGATGGGAC ACTGGGGGCTGGAGGTGAAAA TTCCTTGCCGCTTCCCCAGAGTTGGTGAGATCACTCCCATGCCCGGCGCGC GCGTCGACACGACAGGGACATACTGGGAATGCGCCCTTGCCGTGGAGGC GGGGACCCGGCAGCGCTACGTATCCAGCATCAACCTGTATCCAGCATCAA CCCGCCAAGTTCACTAACTTGGTAGGGGTGAGGTTAGGGATCCTTAGGAG CCCAGGCAGCCAGACTTTCTGGGGAGCCCATTCCCATTTGTGTTGCCAAA GTACCCCCAGCAGGTTGTGGGAATGTTGCCTGTGAAGAGAGTCTGTTGGG GTGAGATCTTGTGTGTGTGCACAGGGTGACAGTTGTGTCCCATTTCCCGGG AAGCTGTGATGGCAGCAGAACCTAGAGGAGCCTGAGAGAGTGTGGGAGA GTGGGCCTCTGGAAGAGTAGAGGCTGCGGAGCCAGGTGCAGGGCTGTCT GTCACCCAAAGGAAGAGGGACTGATGACTCACTGAGCGTGTGTGTCCCCT GGTGGCAGCAGGCCCCATAGTGAACATACCATACCTTTTCTGTCCTGAGC GATGCTCCCAGCAGTCCTGGGAGATGGAACGGTCCTTATTCGGCTCACAG GAAGGACCGCCTTAACTGGACAGACACAGCAAGGTGCTAAAGATGCCTTC CATCAGAGGCCAGGTTGGAAGCTCTAAAGAGACTTCTCTTGCTGTTCTCTC ACCCACCCCCAGGTTGTGTGTGTCCCGCTGTGGATTCTCATGTCCTTTCTG TGCCTGGTGGTCCTCTACTACATTGTGTGGTCCGTCTTGTTCTTGCGCTCTA TGGATGTGATTGCGGACAGCGCAGGACACACATAACCATGGCCCTGAGCT GGATGACCATCGTCGTGCCCCTTCTTACATTTGAGGTAAGCGTTCCACGGG AAGCCTCTTCAGCCCCTGAAGCTTGCGCTTCCCCTGACAGGATTCTGCACC CCTAGAAAGGCAGCCTCTGTCCCTCGAGCTCACAGTGAGCCCACTCCAGG AGAGGGGAGAGAACACAGCCATCTCCGAGAGGGAGCTTCGGTGAAAGGA GAGCATCCTTCCTTTCTCTTGGGGGCAGCACGTGGGGCTGGCAGGGAGAA GAGTGCACCTTTTTAGCCATGGTGCCTCTGTATGGCTCCAGTTTCCACTCT GGGGAAAGCAGAGTGGGATGTCAGATTTGTGTATTGGAGTCACGTGGAGA ATTCTAGAATGGGAGCTGTTGACTCCTTAGAACAAACACCCGGAGGAGTT TGCCATAAAACTGCTGGCACTGGGAACTTTTCAAGTGGATAGGCTATTGC CGAGCTCTGAAGAGGGACATAAAAGCTCATTTCGAGCTTTCCCCAGGGAT AGGTGGTTTCCTGCCTTTTTCTGGCGGTGCTGATGTTCCCTCTTGTGGGAG CTCACGCGGGGGTGGGGTGGTGGGGAGGAACTGCCTAATGAAGTCTGGCT TCCGCCTCTGCCC ATTTTCGGTGCTGGC ATC A ACCGGG ACTATGTCTCTTT CTTTAGATTCTGCTGGTTCACAAACTGGATGGCCACAACGCCTTCTCCTGC ATCCCGATCTTTGTCCCCCTTTGGCTCTCGTTGATCACGCTGATGGCAACC ACATTTGGACAGAAGGGAGGAAACCACTGTATGTACTCAGCATTTCAGAA GTCCTTGGTGTGTGTCTGGGGGGGGACCAGGGGGTGGGGGGTGGCGGATA GAAGTCTAGGAAGGGATGAGTCCCCGAGGGCCCCAATTTAGAAGCTTGTG TGGGAAAGTGAGGGCTGAGGAAATTCTGGGACCTTCTAAGGGAAGGGCA TGCCGTAACTCTGGTGTTCTGCTGGCCTGCACCGGGACTTTTCTCGCAGTG CACGCTGCCATTTGAGGTAGAACCAGACACGGCAGGCAACCTCTCAGAGA TCCCGTTCCCTCCTCTGCAAAATGGGGATCAAGACAGATTCTTCCCAGGCC CGGGAGGGTTTGATGGAAAATCCACATCTCCCACCCAAACCTGGGATTCA TCCTAGGTCCCTGTTGGCCGCTCTGCCTCCCCCATATCCTTGCTGCCATCA CCCGAGTCTTGCCTGTCTTGCCTTGCTAACACTCTATTCCCCTCCACCTGCT TGCTGAGGCAGACACTTCCAAAACGATCTCTGCAGAGGGTGCCTTCCTGG CAAGGCTGTGGGCTCCATGGCACGGAAGCCCAGAGCATTGCCCTTCGGAA AGCCAGTGGGTTTGGGGGCAGGGCCTCACTGCAGCCCAGCAGCCCGGGCT GTGCTTGCTGTTTGTGCCTCTGCCCCCTACCCCGCACCCGGGAGCAGGGA GGGCTTGCACCGAGCTGACACTCCAGTAGCCTACAGAGAGGAGTAGTGG GACTGGGAAAGTGGCTTTAAGGTGGCTCCATGAGTTCAGGCCCCCTCCTG GCCAACCCGTGCATGACTACCGCCCTCACGGATTCCAGAGGGTGACAGAA ATCTTGTTCTTGGGTGGCACTGTCATCCATGAGTTTATCCTGGCTGGAGAA GATTAGCGGAAGACACCGTAGTCTGCGCACCACAGATATTTTGAGACTCA CTGGAGCAGTAGTTCTCAAATTTGGGCATCCAGCAGAATCCCAAAAGGGC CAGGAAAAGGGGACCGCTGGAGCCCACCCTAGCCCGACTCAGTTTCTGGA GGTCTGGGCTGGGGCCCGAGAATGGCATCCCTAACTAGGCCCCGTGGACG CTGTCCCTGCCGGTCCGGG A ACCCC ACTCC A AGC ACC AC AG AGCT AGC AT TTGCACTTCTTCCCCATTTTGGGTACTCAAGCCCTGTTCAGGCTTTGTGACT CAGGAGTCTGGATAAAGTATGTTATGACATTGTAGGAGTGAAACTTCTTG TTACGGAAAGAAAGTTAACAGGAAGGTCAGTTGAGCCTCGTGTGTGAAAT AAAAAATTCTTATTTTTCAGGGTGGTTTGGTATCCGCAAAGATTTCTGTCA GTTTCTGCTTGAAATCTTCCCATTTCTACGAGA ATATGGAAAC ATTTCCTA TGATCTCCATCACGAAGATAATGAAGAAACCGAAGAGACCCCAGTTCCGG AGCCCCCTAAAATCGCACCCATGTTTCGAAAGAAGGCCAGGGTGGTCATT ACCCAGAGCCCTGGGAAGTATGTGCTCCCACCTCCCAAATTAAATATCGA AATGCCAGATTAGATGCCACTTCCGGGGACAGAGCTTAAGTGGACTGGGA CGCACTCTCTCCGCCTTCCTCTGCCCCCTCGTTCACCCCGCAGACCAGAAC CAGTACTGGAGCTGGGTCTCCAGGTACGTCCATCTCATGCCTTGTTTGCAT CCAGCGCCTATCAGCCACTCACCACGACGGGACGCGGAAGTGGCAGGTG ACGGGGGTGTGTGCCAGCAGATGCGGATGCCAGGAAGAGTGTGAGAACA GGGGTGGGATTACCGTCTGTCTGGGAGGGGCTCCAGGTACCCCTCTTCCC CGTCAGACCCACTGGGAGATGGCTGCTTGCCAGGCCCCCAGAAGGAACAT CTGTCTATACGGTGCTGAAATCCCAATCAAAAGTATTGTTTAGAAATGTAT TTCTCCACAGGGCTGACCTCCTGCAGCTCGCTGAGCACTCCCAGGTCCTCA GCACTCCCAGGTCGTGGCTGGGGCAGTCAGTAGGAACTGTAACTATGTCT CTGATGCACCACGTGTTTAGACACAGCACAGTCCTTTTTTCTGTTCCTACT GTGGAAGTAGTTTCTCTTTGGGCATGCTGACAGCAGTTTTTCATAGCCTCA CGGATGAGCCCTTTCTACGGGAGTGACTCCATGCTTGTATACAGAGTATTT ATACAAATGTTTTAGCATCTTCATATGCGGTGTTAACCCCTAGTTCTGTAC AGCATATTCTGTTCAAGTATTTTTTTACAAGCTTGTGCTGTAGGCACATGC CTTCTGCTGCAGAAGTGGACGCCCGTGGCACACTCCCCCCCCCCCCCCGT GGGGTGCCACGCCTTCATGGGACATTGCCACTTCTGCCCTGGAACTCGTG CAGGTACGTAGTAGCTGCTACTGCCACAACGGCAACACCAAGCAAGAGA TGGTCCATGCTTTTCTGACGTTCTCAGAATAGTGGCTAGCTTCAAACCTGA CAAGCGCTGCTTGAAGCCGGAACACTAGAGAATGTTGCTGAGAGCAGAA ACGGCCACGCGGGTCACGACTATGCGTGGGAAAGTCTCAAGCTTCCCTCC TGCC AGC AAC A AGA AGGCTTTGG AGTAGGC ATG ATGTTTTC ACGTGTGCG TGCCGTTTCTCCAAGCACTGCAGGTTCCACCGTGTGTCAGAGGCTGCAAG TTTAACATCCTCCTGCCTGAAAACAAATAGGTCCTTTGCTGAAAAGAGGG TAAAAAAAGAGCTTTGATCTTCTCAGCCAGGAGAAGAGGGTGGTGTTTTC ACGCGGGCAACTGCTCGCCGGCCTACATGGGGTTAATTCAAGTCTGCTGC GAGC ACGACTCCGCCCTTGGCACTGGCCTCC AGC AAGCCCTGTTCTCTTTG GGGTACAGGGGAACGGGATGGTTTAGACTTTCCTGCTCAGTGTGTAAAAA ATGTAGCTAAAGCCACTATTTTTGCTCTCCTTAAGCTGTTCAATAAACCGG TTCCTCATTTTACACGTGCATGATGTGTATCTTCTTTGCTGGATGGGCCAG GAAACTGGAGTGGTCCTCTCAGCCAGCCTCAGAGGAAAGAAATCTCTAGC TGGCACAGGCAGCCAGTGAGTGAGGCTGGCGGCTGCAGGGGCACAGCCT TTAGAATGAGTCCTTCAGTGCACAGGTCCCAGGGTATACGGGGTAGTGGG AGGAAGGAGGGGACGCCTCGCAGATGCCACTGTTGGCTGGGCTACACCTT GCCACACTTGTTACTGCTTAGGAGGCTTTCTGGAGTGTTCCTTGGGTGCTA CGACAATCTGCAGCAGACACTGTCCTTTCACCGCTCCTGGTCCTCGTTTGC TCCCCAGTGATGTCAACAGCTGAGGACTGCTCACGCTGCAACAAAAGGCT CTGCAGTCGCTGTCTAGCTTGCCCTAGTCGTCTCTAGAGTTCTGCCTGAAC TGAAACTCAAGTGGGGTTCAGCTCATGACTTGTGGCAATTGACCAGGAAA TTCACCAGTTGCTGTGGCTGGAAGGATTTTCAGTCCTGTGGGTTGTAACCA GAGGCCACAGGTGGATTCTGCCTTAGGCTCATGAGATTTCCGACTTGCTGT TGAAGAAAATGCCTTGTGAAGTGACAACAGTAGCTCTGACCCAACTGCCG GTGCCTCGCTAGTTCCTATACGTCCCACTGGATCCTCACAGCCCCGGGAA GCAGGTGCTACTACTCTTATCCCCGGGAGGAGACAGAGGCCGAGAGAGG TTAAGTGACGTGCCCAAGTCACACAGCTCGGCAGCGGCCGGGTTGAGCAT CAGCAGTCTGTTTGCAGACCCCTCACTGTCACCCCCTGAGCCAGTGCGCCT TGGGCCCTGCGGTCAGGATGTCTCAAGCGTGGAGGCATCACCGGTTCGTG GCAGTCTCTGGAAGGTCACTGAGCTCTGTGCCCAGAATCGAGTCGGGGGA GTCTGTGCAGAGGTGGCCCTGTGTGTGGGGACAGTGTGTGACACAGACAC TGCTTTGGATGGACACCTCTCCCGTGACCTCCTAGCATCCAATCCCAAAGG AACAACTGTTGCAGAGATGGACCGCTGGACACAAACCCACGTGCGTTTCT CTGG AGAC ACTGGCC A AGG A AA AC A A A AC ATGCTCG A AGGCC AAC AGCT GCATGCCCCACCGCGATGTGACCGCAGACACCCGGGGTGTAGAAGGGTCT CTGCCTGGTGGGGGGACACGTGCAGGCCGAGGAGAGGCAGGAAGGAGGC TGCCTCCGACTCCCCACTGGACTGCATGGCGACGGCGTGTGGTGGGGCAG TCAGCTAAGCCATTTGCCTAAGGGGCTGTCGGGCATCTGCGTGCTGGGGA CCG AC AGTGTGGGTGTGTTAGGAGG ATCTGTATGG AGC AC ATTGCTGCCT CTGGCTAGGACAGGGTGGAAAGGGTGGCGTGGCTACAGCCTGACCCATG GGCACCGTCCTACCCTTTGTTCTGTGCTTCCGAGTGTCAGTCATGTGCTGG GGTCTGTGGGCCCATGACTCAGACGGTGAGCTCTGACCTTCCTGAGCCAG GGCTTTGCTGTAGTTGTGCCTGGCTCAGGAGCTCTAGGACAAGGGGACCG CTCCAGGTCTGCATCTACGGTGTGGCAGGGCCCCTCGGCACTCTTGTGCAC TAGTGTCATCTTTCCCATTGAAATGACTGTGAGGACCAGAATGTGCACAT GCAGATGGGCAGCTACTTGTCTGCCTTGGCCCTTTATTACACAACTTGCTG GGGGTGGAGATGCCACCCCCCGGCAGTCAGAGCCCCTTTATGATGTCATG GGGCTGGTTACATGACTGCCAAGGGGTGCTGCTGGCCACACTGCACTAGC AAGTTTGCCAGATGGAGGACAAGCGATCATTGAGTATGGCTCGCTGTGAA GAAAGAAATTCGAGAGGACAGGATCATGGCTTGGAAAGGGTGCCTTTCCC TCCCCAGTTGCAGTCAGAGACCTACCTTCACCCAGCAGATCCTTCCCCTGC CTGGGACGACCCGGGGTCCACTGGGAGCCCTAACTTGAGGCTGCTGACAG AAGAAATCGCTTTCCAACCTCTGGCCGAGGAAGCTTCGTTCAGAAGGCCG CACCCTGACGGTGACGTCCCGCCCCAGGGAGAAGATAATCTCCTCTCCCT CCCCTTTCCACAGAAACTGTGGAGACTGGTCAGCAGCAACCAGTTTTCGT CCATCTGGTGGGATGACAGTGGGGCTTGTAGAGTGATCAATCAAAAACTC TTTGAAAAGGAGATTCTCAAAAGGGACGTCGCACACAAAGTGTTTGCCAC AACTTCGATAAAGAGCTTCTTCCGCCAGCTAAACTTGTATGGCTTCCGAA AACGGCGTCAATGCACTTTCAGGACCTTCACCCGCATTTTCTCCGCAAAA AGGCTGGTCTCCATCTTGAATAAGGTAATGAACGACAAGCCTCTGGAGGG GTTAAGTCGGTGGGCTCTGGGGCCTGGTCGGGTGGAAGTCCCAGGACTGC CTCCTGGGAAGTGGGCGACCTCAGGCAGGGTGTGGGGCCATCGCTGTGGG CCTGTGTCCCCCTCTGGGTGGAGGTGACATGAACTAAGAGTGAATGTGGG GAG AGGGCTGAGGATGGTGCGGGCCCCTCTCG AGTGTGTAAAAT ATC AC A GGTGCCAAGTAGCCGTATCTGCGTGTCGTCCTCCCCGGGGCCAGCCATGT CATCTGGTGGTTGCTGTGTCCCCCTGACTCCACAGCACATTACCCTGTGAG GTGAGCAGGCCAGGGGAGTCTGGTATTTGTACCACTGTCACCCTAGCTGG TGTCTGGAGAGGTGCTCAAGTGGAAGCACTGAAGGGCGCCTGGCGCAGG AGGTGCAGATGCTCCTGCTGCCCTTGGTAGGTGGGCCCCTGGTGTGGAAG AGCCAGTACCCAGGGCCTCCAACCCAGCCGGGGTGCATTCTGTTGCCAGC TGACACTGCATGGGGGAGGCCCAGAATCTTCTTCCCTCCTGGTCTGCAACT TCAAAGACCCTTTCCGCCGGCCATGGACACCCTAATCTGCCATTTTGAGGC TTTTTCCAAGACGGAAAGGCCCGCCACAACTTGGTAAACCTTGACGATGT GAACGCGAGTCCCCAGCTTCCTTTGGGGACTGGGACCTTTTCCAGAAAGG CCTCCTGGGCCAGTAGAGTTCTCTTGCACAGGGGCGTAGATGGTTGGTAG TTGTAGTCCATCCTTGTGACTTGCAGTTTCCTTTTGTTTATTTAAACTTTGA CTTATAGTTAGAGTTCTACTGCCATCCTTACTTTCAAAGAGACTCCCCTCA CCTCCTCGTGAGGATGAAGAGAAGAGTGGGTGTCAAGTCTGCACCAAGAC ATCAGGAGGAGGACAAGCCAGAAGCTGCTGGATCCTGTCTGGCACCAGC AGACACTGAGCAACAAGATCACACGTCTCCGAATGAGAATGACCAGGTC ACACCGCAACACCGGGAACCGGCCGGTCCCAACACCCAAATCAGGAGTG GCTCTGCTCCACCAGCAACTCCTGTGATGGTGCCTGATTCCGCCGTGGCGA GTGACAACAGTCCAGTGACCCAGCCGGCCGGCGAGTGGTCAGAGGGCAG CCAGGCTCACGTCACTCCGGTGGCCGCTGTCCCTGGGCCTGCAGCGCTGC CCTTCCTCTATGTCCCTGGATCTCCCACTCAGATGAATTCTTACGGGCCTG TGGTGGCCCTTCCCACAGCGTCCCGTAGTACCCTTGCCATGGACACCACA GGACTTCCTGCACCTGGCATGCTGCCCTTTTGCCATCTCTGGGTACCGGTG ACCCTAGTGGCTGCTGGGGCTGCACAGCCTGCTGCCTCCATGGTCATGTTC CCCCATCTCCCAGCTCTGCACCACCATTGCCCCCACAGCCACCGCACGTC ACAGTACATGCCAGCTAGCGATGGGCCCCAGGCGTACCCAGACTACGCAG ACCAGAGCACATAGAGGGCAGCATTTGGGCAGAATATGTGCTGGTCAATA AATGTGTCAGAAAATGAGTAATTTTCTGACTGCACAAAAAAGTCTTCATG GTCTCCATCCTTTTTCTTTCTTTGCAGTCCAGTACCCCCCTCCGACATCTTT GGCTTGCATGATAGAGACTTCAGCCTAACACGCCTCCCTGGAAAAGTGCC CAGGAGATACTCGAGCAGGTGCTGGGTTTTAGGAAGCCCCTCAGACATCC CGCCTCTACTCATTAAAAATTACCTGGAGCTGGCCGGGCGCAGTGGCTCA TGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCACGAGCT CGGGAGATCGAGACTGTCCTGGCTAACACGGTGAAACCCCGTCTGTACTA A AA ATAC AA A A AATT AGCCGGGCGTGGTGGCGGGC ACCTGTAGTCCC AG CTACTCGGGAGGCTGAGGCAGGAGAATGGCATGAACCCGGGAGGCGGAC GTGCAGCAAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAG CAGGACTCCGTCTAAAAAAAAAAAAAAAAAAAAAAAATTACCTGGAGTT GCTCGCTGAGTGCTAATTGTGCTGCTAGCATTGCCGGCCTAGGGTTCACA ATGACTCTGTGTTACTGGCCATTCCTTGTCATGGCGGTGCCTTTATATAAC AGGATCTTCACTTTACATACAGATGAGATCACGTCTGGCTTTGTCATTGGG TTAAAAAGTGCCCTTGATAGTTGTGGAGGTGACACATACTACCGTCGACT CGAATAGATCTGCTAGGCGCCAAGCTTGTCAGCCTTACCAGTAAAAAAGA AAACCTATTAAAAAAACACCACTCGACACGGCACCAGCTCAATCAGTCAC AGTGTAAAAAAGGGCCAAGTGCAGAGCGAGTATATATAGGACTAAAAAA TGACGTAACGGTTAAAGTCCACAAAAAACACCCAGAAAACCGCACGCGA ACCTACGCCCAGAAACGAAAGCCAAAAAACCCACAACTTCCTCAAATCGT CACTTCCGTTTTCCCACGTTACGTAACTTCCCATTTTAAGAAAACTACAAT TCCCAACACATACAAGTTACTCCGCCCTAAAACCTACGTCACCCGCCCCG TTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTG GCTTCAATCCAAAATAAGGTATATTATTGATGATGTTT

SEP. ID. NP. 17 Helper dependent vector C4AFO

The sequence is shown without transgenes.

AAACATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATG AGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTG ACGTAGTAGTGTGGCGG A AGTGTG ATGTTGC A AGTGTGGCGG A AC AC ATG TAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACA CAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTT GGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGA GGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTC TAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTT TTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTGATTCGGCC GCTTGGGTCATAGACTTCTTTGAGAACCAGTTATAAGCTATGGTTTCTCTC CACAGAAAAAGCACTTATGGTGTCTCCCCCTTTCCAGCCCACCAACATTTT ACATCTAATTTGGGGGGGTTTTCTGGACCACTTAATACCCATCCATGGATC TCATGTGAAGACTCCCCTGGCTTGAGAAATCACTGTCTTGTTGAAAATGG GAACAAAGCTAAGTCAGATAGCTGGTTCATCAGCAATGACTTTGACCAAG CCTGATCCCACCCTACCCCACCCCACCCCAGTGACCACCCCCCACAATGG AGCACACAACTCTAAACTGGTTTGTAGGTATGTGTGTGTGAACACGCTGA GGAATCTGCAAAACCAAATGGTGAGTGCAAAACCAAACAGTCACGAGTA AATCTCACAACAACCACGTCCTGAGCTGCAGCCCTTGTTGAACTATACCC CACTAGGGCCCCAAGATTTTAGGACTTGTGTGTGGGTGGGACCTCCCCTTT CTATCATGCTTTAGAAGACAGGGATTTCACCAGAATTGAACATATTGAAC ATATGACCCATTTTTTTCAGCCAAAGGCAATTAAAATAACTTCATACTTGA TATCCATGTCAGCAAAAGCTGCAAAACGCAAATGGGTGGCTGCTAAGAGC CCTGGTACCCTGACGAGCACACCAAGTGCTTAGCAACAGTGGTGTCCAAA GGACCAGCTGGAAGCCTGCCTTGATGAGAAGTTGCTCTTCTTTCTACATGA AGGAACACCTCTACTCTCCTGCTTTTAATACCTGAGCTGTGAGTGATCATC TATGTCCATTAGCAAACATCCCAGTGGAGAAGGAAACACTCATACCCGAA ATCTAAGCTACATAGTTGGAATCACTTCAACTTATTGCAATAAACACTTAC TAAGCACCTATTGTGGGCAAGTCTTTGCAATGGATAATAGTTCAGTAGAT ATTTTGATGTAATATTTGAAATAACAATAAAAATTGCCACCACTGAATTTA TTGAGCATTTGCTGTGCTTTAGGCACTAACCCAGGTTCTTTAAATATTTGG TCTTATTCGATCTGTATAAATAGCCATCTATGAGAAAGGGACTATTATTGC CCTTATTTTACAAATGAGGCCAATGAGGCCCAGAGAGGTTAACTAAGTTG CCCAAAATCATACAGCCCACTAGTGGCAGAGCAGGATGCAAACCCAGGC TTGCCTCGTTCCC AAGCCC AC ATGTCGTTTGC ATTGGTGTGG AGGTGTGC A TGTGTTTAGTCATTAGCATGTTATATGATAAGCAAGTTTTGAAACATAGAA ACTTAAAATGTGCCATTAAGAAAAGTACAGGCAAGGTTTTCCAAGGGGAG GTGTGGACCTCCGGACAAATTTTTAAGAACTAATTATAAATACTTAAAAA TGGGAATAAGAAGACAACCTAACTACCTGAACAGTTTTAGAGATGACTCA TGCCC ACCCTCTAAAACCCAAAC AAAAAC AAC AAAGTC AAGAAAACCCA TGAAATCTTAGCAAGCGATTTCTATGTACTTGTGAAAAGGATTTCTTTACC ATTCTAATGGGATTTATGCCAACCATAGAGGGCTCAGTGCCCCTCCCATG GGGTGGTTAGTGAGTACAGAGCTGAGCTCACCGGCCATCTGCAGCTTCAT GTTATCAAGCTCCAGTTTGTCCTTGGAGCAAGGTTATCTGGGACATGAGC AGAGGCATTGCTTTCTGCAATGGACAGTTCTTTCTGCCTGCATACCTAGCT CCTTGATAACTTTAAATACCATTTTATAGCCACACTGGAGTTTTGAAGACC TCAATATGCAAATATTACTCAGGTTCTGATTACTTGTCTGCTCCATGATAA CACACTCTAAAAGCAATGAATGGGGCTTATTTGTAGAGAACTGAAGCATT TTAAGCTTTTGCTCAGGAATCCCTGGTAGCTTCCTGTGACTTGCAAGATAT TAGTGATGGGTCAAGAAACAGGACCCCCCATCAGCATAACATACGCAGTG CCTCAGTAGTCCATCAGGCAGAAAAAACTGCAGATGGCACATGGAAATG ACCAGCGGCGGAAGATACCCCGACAGTGTGGGCAGTTCTATTTCAGCAGC AATCAAGAGGGGGCCTGGAGCCACTCAATCAAGTGGAGCAGGATGGGAG CAAGCACTGTGCAGACCAATGCAATCCCCAGTTAACACAAAAAATAAATA AAAGAGATGAGATTCAGTCTCTTGACTGTGACTGACTGGGAGCTTTATAG CTGATGCTTGTGTCTTTTCTCCATTTTATTTAATTAGGAAAAGAAATGCTT ATCACACACTCTACGTGTGAGGTACAACCTCCACAGGAAAGGTTGTTAGG AACATTTCAACTTCTAGAAGTTTCTAAACATAAGGTAAATCCATCTTTGTC CTTGGGATCACTGCACATCTCAGAAAGGCAAATAAATCAGTAATTGGTGG GCATAATTACTAGCTCATGGACTGACAAGGTCTACACTATTTCGAATCTCA CAGAAGTAAGCCATGGGACAGATAGAGTCTGATAGTGGTGCCCCGTTTCC TGGAGGTCACACTTACTCATCCCCCTGGACCCTGGGCTTCTCATGATTGTC AGAGAGTTTGCTGGAACCAGGTCAGCCCAGTTTCCCTTCCCCTGAAAAAT CCTCCAATGGCTCGCACCAAGACTAGAGATGCAAGTGCAAGCACATCCAC CCTCTCAGCAGCCAGGTTTTGCGTTCCATAATGTCACGTACCCCCAGTCAT ACCAATCTCCTTGGAGCTCTCCAGACAGGCTGCCATGTGTGGTCGGCCCTC TGTGCTTGTGCTCCTTGGTTTGCCAAGCCTGGAATGCCCTTTCTCCATGAT TGTACTCTGGGAATTCTGTGTGTCTTTCGAGATGAAGCTCCTCCACCCTGG AAATTCTGCCTCTCTTTCCAGGTCCAGCTCCTGTGGCTGAATACCCTTGGC TG ACTGA AC ATTCTTGCTGGGCC AAGTCTTAATC ATCTCTA A ATCCCTCTG GTGCCCCGACAGTATCTAGCCCAGGCAAAGGGCTCAGCAAATACTTGCAA AATTCAATAAACTTTACTATGTTACTAGCTTGGCATGATGTTCTAGGCACT TGGGAGATTATAATCTGGTGAGCATTGTGCTAGTCTTTTTTTTGGGATAAA CACCAATGATAAACTGAGCCTTTACTCTCATTTTAGCACATCTACTCATCT CTTTTCAATGCTGGTGGGTTTATAAAACCCATCAGTAGAACAGAGAGTGA TCATAATCATATGGTGAAAATAACATCAGCTAACATATTTACTGAACATG CTTTAGTGTGCTGGGCACTGAACTCTGTGCACATGTGAATTTGAGATAGAT TGTTCCTAGCTAATAAGATGAGGAAGTGGAGATGGGCTTACTCAAGTCAC ACAATAGCAAGATGGGGTACAGACAGAAACCAAGCTAGAAACCAAGCAA CCCCTGGGTTTGGAAATGCATGGGCTCCTCCTCTGCACATGGCGAGGAGC AGTCAGGTGCTCCTCCTTCTCTCATACTGAAATAACTCTGCACTTTTGGCT ATTCTGTGACACTCTGTGCTATTCTGTAGCTCAAATGGCTCTGGTGCAAGG AGCTCAGATTATGACTAACCCCCATAGATATTCAGCTGCTTTGCAAGAAG TGGATGAATGCTCTGGCTTTATAAATTATTGACTAGATTGGATATTGGCCC AATCTCCACTCTGGTGATTCGGAAGGAGGCATATGCACATTTGCAAGGTT ATAGTAGTGCACTCTAATTCCACTGGCTTCTGAGAGCTTGTAGGTTTCTTG ACTTAATCATTCTGGAATTAAGGTAATGGATCCTCAACACCTTTTCTTTCC CTGGCCTTTACTAACCATGTAACAGAAATGAGCAGAGAAAACCCAAGAA AGCGAACTGGAGACTTGATGAGTGTGTCAAAGATGCTCAGAGTCCAAGGT CCTCTGTGGCTCACTGACTTCAGAAGCCAACCTCCGTTGTTCAAGTCACTT GTGAGGTTACTATGCTAGAGCACTAAATATTATCCAGATAGCCCAAAGAG GTGAAGGCAGACATGTGGAAGAACCTGGATTTTTGGGCAAGTTGATGAAT GCCTCCTGCCCCATATCAAAGAGAGGTGATAGGAGACAACTTTGTGAATT TGAAATAATGCACCGGGGAAACAGGAAAACGTAATGTAAGTCACGCTTCT TGGCTTTTTTCTTGCC ATT ACCCT ACTTGGCC A AGTGC A A ATGGG ATTTC A ATATATCATAAGTATGCATCTATTAATAACAATGCAAGAAAGCTGTACAA CTGAAGTCTGAGATTTTGTAAGAAACAGAATCTCTGTAAGCATCACCATC CAACAGAACTTCCTGAGTTGATGCTGAATCATCCCAGAAAGAAGGCGCGC CAGCTCTGCAGGATCTTCAAGTCTGGGGTGCCACCAGCAAGCGACGGTCC TCC ATGGGCTCTTC ACCTTACGGC AGTGTCC AG AGGC ACCGCC AGTCCTCT GCTCCTATGCTGGTCCTGCTGTCCCTGGCAAAAGGAGCCAGAGCATTCTCT CCAGGCCTCCCGAGGAGGCTGCTTCCTTTGTTTTGCAGATGGAGGCTCCCA TCCTTTGTTCTGAATCAATGTGCTCCAAAGATAAGCCCCAAGAAAACAGT TGTTGCCTTTTGACACTGACAATTAGAATCGTTGGAAAATGGAGAAAACA GGAAATGGCAAATGGTTTCAGTGACCAGGAGGAAACCGTGCCTGAAAGTT GCTGCTTAGTGACTGGGACACTCGCTTTCTGCTCTCTTATGAAGGACAGCC TAGGCCGTGTGGCCTTTTATAAACAAAGCTATGAAGGGGTCGTCAAATTT TCTAGGGCTGCAACTGTGGCACTACGTCCTGTTGTGCCAGGTGACACTGA CAAGCAGCACTGAGTTCTATGCAAGCCCAGGTGTGCTTCTCTCATGGTGA CCCCCAGAGAACTAAGGCCCAGCTCTTCCTCTGTCACACCCCTCCCAGCC CCCACTGTCAGACAAGGGACCACATTCACAGACAGTCTCAGCCAAGATGG CAACCTTGGAAGTCCTGGGGATGCCTTTCTAGAAGCTTTAGGCTGACGCC AGGGAGCTGTAAAGCCCCCACCTGTCCCTGGGGGTTGTGTGCTGGGCAGG AGAGAGGGGAAAGAGCCCAAACTCCACCACTGCCTGCTGGCACAACTGA GCCCACGCCAGGCACTGCACCAGCTTCACTCACCAGCTACCACATGCTAA TGTCTCCTGTTTACCCAGGTGCAATCACCTGAGTGCTGCTTTCTCACCACT GCATGGGAAGAACGATCTCATTACCAATTCAACCACTTAAGCAGGCAAAA GGACTCCAACGGGGGAAAGGCAGAGGACAAACGATTCGGGGACTGAGAC AGATGCCCACAAAATGTTCCTGGATTTTAGCCGGATTCGGGCTGAAGTTC CTTGCCCTATGAGCCCTTGGAGGAGAGCATCCTGACTCAGACAAGAATTC AAATGACAAGACTTCTGGGGGATGGCAGTGAATGGATCCAAAGCCTTGTT TTATAAAGAATGTGGAGGAAGGGAGGAAGGAAGGGAGGGAAGGGGAGA CTGAGAGGGAGAGAGAATAAAGAAGAGGAAGAGAAGGAAGAGGAGGAG GAGGAAGGGGAGGGGAGAGGAAAAAGGGGAGGGTAGAGGAGGAGGGAG A AGGGGA AGGG AGG AC AG A AG AGGGG AGGGAGCGGGG AG ATTATCTAC CCCTCCATGCTAGGGAACTCCTGTCTCCTTTTCCAGCAGACACTGCTCCAT GAGTGCCCCCACAGAGAGCCAGCCTGCCACACAGCAGAAGGTGCTCAGC TATCTGTCCCAAAGGTTGAGCACTAGTGTATACCACAAGGAAAAAAACCT TCTTTTGTACTGTTTGGAAGCCAGAATTAGAAACAACTCGTTCACCCTTCT GACTTTCCCTTTGC AATGAC AGG A AGAT AGTATACTGC ATGGGACC ACCA CACATTCAAGATCTGAGTTGTGGGGACCCCCACCCCCACATGGGTGGAGC CCCCTGAGACCCGTGGGCTCTGACTGGCCATGGCCCATAGATAGTGACAA CATACACACTGCTATCTGAAGCCTAGTTGGAGGACGGATGGATGCTTTTA GGAAAAAATTCCCTTTAGTCGTCCCTCAGAAACTACAACGGCTTCCTAAA ATGAATGGCAGGCAAGTTTTGATTGTTTCCTAAGTTCCCCCTAACTCTCAG AGCCTGTGACTCAGCTTCTCCAGAATGCCTGCAGGGAGGCAGGACCACTG GGAGTAATGAGCACGAATTGGGCCACTTCTCCCAGTGGACTGTTAACGCC GAGATCTGGGGTGGAACCCACTGAACACCTGCCCTTTTGCGTTGCCCAGA ATGCAGCCAGAGGGATACGGGCCACAGTAACTCTGGGGCCCCTGAACCTA CCTTCCCAAGACCCTGACGGGAAGGTATTGCTTACATGACAGTCCAGTCA GTCCCTCTGTCACTCAGTTAACAAACATGGTCATGGGTCTACTATGGGCCA GGCTCTGTTCCGGGCATGGGGATAACACCGTGTGTCTCTACTCTTATGGAC ACAAGTAAATCAATCAAAGCAGATAATGTTCTAATGACCACCCAGTAGGG TGAAGGGATGGAGGGTGGGCAGGTGCTGGCAGAAACCTAAGGTCAAGTC ATGGGACCCCAATGGGGAACTGGAAACTGCCACACCATTTTCTATGTTAG GGGTCTTTCCTTGCCCTTCTAGAGACCTCACCCATGGTAATGATGCTGCCC TCAGGTTTGAACTGGTCTGAAGCTGTCACTGGGGGCTTCACAAAATGAAT GCCACTGAAAGCAAACGGCAGAGATCTGTATATGAAATTCTTTTTACCCT TTGTTTTCTGTCTTCCCACATGATCTATGACTGCTGCAAACTCTATTCTGAC TCCTCCTCAGTCTTGGCCGGTCTGGGAAGAACTTGGTTTAGATGCTGGGA ATGATCCCGGGCAAAGGAAACAAATATATTTCCAGCTTGCTTTTCTTCTTC CCCTGTGGTGCCTGCATCCTCTGTCTCGCACTGACTCACAGGCTGGACTGA CAAAGTGGCTGTGTTGACAGGGAGGATTTAGTGGATGCCAGGCCTGACCC TCAAAGGCCCTCAGGTGGGGCCAATTTCCAAGGTTCTTAGAGAAGATTTG GGGCCACAAATCTGCTCTGCCACTTCACTGCTGGGTGAAAGCTGTTGGCTT GAGAAGAGAGAGAATAGGTGAGGGACAATAATTCTCAAGGCAAGCTAGC TGATATCCTTTCATTTGTGGCTTCCAAAGACAGGGAAAGGCAAGGCAGAC CAGGGTGGAAACAGCGACATCCTGAGGGGACGACGTCCGTTTTGCTAATT CTGTTTCCCTGGCAGCTTTGTGCTGGACAAGATTTCTCTGGTTTCAGCTGA AG AGTG A AC AGCTGAGGAGCTCTGTGACTCTG AGAGAG AGAGA AATC A A TAGCCCTTGAAAGGAAAGCACTGCTTGTTCTTCCAGGCCAGGGCAGGCAG GATAAAGAGAACAAGGCTGTACATACCAAAGCCGAATTTCCATTAAAGG CCAGAATCCTGGGAGGCCCTTTCAGCTTAATGACCATCTTCTGATCACTAT CACTTAGAAAATGTTCATGTCTCCAGTTTTCTCTGCAGATAGTGCAGACAT CCTGTGCACATTGATATTTTATGCACCAGATGCAATTCTCTGCAAAGGTCC CCTCTGGCTGGTGTCCATCCGCTTGCTCTCATGACCACTGATGCGACTCTG GGGGCAAGTCAAGCCATTCCTTCTAGACTCTAGAATCTAGAGGATGATCC CAACTCACAGGCCTCTTGGACAAGCTTGCTTTGGATTCCTTCTGCCTGTAC TACCCAGACTGCCAGTCCTCCAGCAGGGCAATCCAACTTCCTGCCTCTAG GGTGTGGGAGTCCTGGGCGGGGTTTGGAATCCACAGCAGATGGCTGGCAG GAGAGGCAGTTCCCGAGCACCCACTAGGTGCCAAGCAGGCAGGTATCAG GTGCAGGGCTTCGGAGGTGCAGAAGCACAGCCCCTGTCCCCCTCAGGTTC CCAGTCTGGCCTCAGGGACAGACAGGCAGGTAGGTATGGAAACGGGGTA CTGGAAGGCATACAGCTGAGGGAGAGAGCAGCTGCACCCTGGTCAATCC CCCACTCATTTCCCTGGAGGTGGGGCTGAGGCACCTGGAGCACTCTGCTT GCTTGGGTTGTCCCTTTGCCAAAGAATGCCGGGCAGAAAGTAGAGAACAA GCAGGCCCAGGAAGCACTAGAAGTCCCTCATGGGTGGACAGAGCTTTACC TTCGGAGCTGTCACCTCCTGTGTTCCTCCCAACAACCCTGTGAGGCAGGCG GGCAGGCTGAGCAGGGGACTGTGCCCCGTTGACAGATGAGGAAAGCGAG GCTCAGGGGGAAGTGACTTACCTGTGGCGTCGCAGCTAGGCAGTAGTGGA GCGGGCACCTGCCTCCAGGCCTTCGGATCCCAGCTGCCCCAGGGCGCTGC CTCTGAGGTGCTGTACGAGGAGGTGACTCGGGCCAGCTCATGAGCAAAGG AGCTGCCGGGCAGGAAGGACTCACTCCCCAGGGCCTGGCAGGACGGGGG CTGTCGCCCGAGGCTGACCTGGGGGTGCCTGGCTTCCCCAAGCTTGCAGG CTTTGTCTACATGAGTCTACAACAACCGATTGAACAATCCATCAGCTGCTT GGCCACAAAATGGTTCTGACTGATCTTGTCACTGCCCTGAGTGTCTAGCTG GGTCATCTGGGTGTTGTGGTTATACCCACCAACTGCTTTGGTGGAACGAA ACCGCGGCCGATCTGAATTCAGATCGGCCGCGGGATCATTCCTGGACTCA GATTGTTCTGAAGAAGCCCAGTTCTGGGTGGCATCAAGTGCTTGCTAGAT GGGGGGCTTGCCTTGATCCGGCTAC ACTTGGAGGTGACTTGTTCTTGGAC GGCTACATACAGAAAGAGAGAAGTGGGGATGAGTTCCAAAGGCATCCTC GACTTCGGCTGTGGCCACCGGAGGGTAGCTCCTGGCCCAACACGGACTTC TCACCTCCCGCCCTTGGCTCTCTACTGAGCTCCCCCCTGCTCCCCAATTCC TCGCCATTCCCCTCATTTCTCTGCCCTCAGCCTGGACTGCAGTTCTTCTGG GAAGCTGCCCCAACTCCCTAGGTCTGTGCTCACCAAGAGCAGATCACACT GGACTGAAATGCCAGCTGATTTGTCTCTTCAAGAAAATTGGAAGCTCCTG GAGGTCAGGGTCCATGTCTGCTTTTACACTCAGTGCTCTGTATGCAGGCCT GGCACTGCCCACCCTTTGACAGGTGGTGCATATTTTGTAGAAGGAAGGAA GGGGCCAGGTGGGGTGGGCTGGGCTGGTGGCGGGAGCTAGCTCAGCCTCT TAGATTCTCTACCCGATGGATGTGACCTGGGACAGCAAGTGAGTGTGGTG AGTGAGTGCAGACGGTGCTTTGTTCCCCTCTTGTCTCATAGCCTAGATGGC CTCTGAGCCCAGATCTGGGGCTCAGACAACATTTGTTCAACTGAACGGTA ATGGGTTTCCTTTCTGAAGGCTGAAATCTGGGAGCTGACATTCTGGACTCC CTGAGTTCTGAAGAGCCTGGGGATGGAGAGACACGGAGCAGAAGATGGA AGGTAGAGTCCCAGGTGCCTAAGATGGGGAATACATCTCCCCTCATTGTC ATGAGAGTCCACTCTAGCTGATATCTACTGTGGCCAATATCTACCGGTACT TTTTTGGGGTGGACACTGAGTCATGCAGCAGTCTTATGGTTTACCCAAGGT CAGGTAGGGGAGACAGTGCAGTCAGAGCACAAGCCCAGTGTGTCTGACC CACCCAAGAATCCATGCTCGTATCTACAAAAATGATTTTTTCTCTTGTAAT GGTGCCTAGGTTCTTTTATTATCATGGCATGTGTATGTTTTTCAACTAGGTT ACAATCTGGCCTTATAAGGTTAACCTCCTGGAGGCCACCAGCCTTCCTGA AACTTGTCTGTGCTGTCCCTGCAACTGGAGTGTGCCTGATGTGGCACTCCA GCCTGGACAAGTGGGACACAGACTCCGCTGTTATCAGGCCCAAAGATGTC TTCCATAAGACCAGAAGAGCAATGGTGTAGAGGTGTCATGGGCTACAATA A AG ATGCTG ACCTCCTGTCTG AGGGC A AGC AGCCTCTTCTGGCCCTC AG A CAAATGCTGAGTGTTCCCAAGACTACCCTCGGCCTGGTCCAATCTCATCCC ACTGGTGCGTAAGGGTTGCTGAACTCATGACTTCTTGGCTAGCCTGCAAC CTCCACGGAGTGGGAACTACATCAGGCATTTTGCTAACTGCTGTATCCTA GGCCAATAAATGTTGATCACATTTATAGCTGCCATGGTAGGGTGGGGACC CCTGCT ATCTATCTGTGG AGGCTCTGGGAGCCCCTG AC AC AA ACTTTCTG A AGCAGAGCCTCCCCAACCCCTTTTCCATTCCCTATACCTGACAGATGGCCC AGGAACCCATTAGAAATGGAAGGTCACTGCAGCAGTATGTGAATGTGCGT GTGGGAGAAGGGCAGGATCAGAGCCCTGGGGGTGTGGCAGCCCCCAAGT GATTCTAATCCAGATCCTAGGGTTGTTTCCCTGTCCCATTGAAATAGCTGC TTTAAGGGGCCTGACTCAGGGAAATCAGTCTCTTGAATTAAGTGGTGATTT TGGAGTCATTTAGACCAGGCCTTCAATTGGGATCCTGCTCTTAGAGTTGGA TGAATTATTTAACTGATTTTCAGATCTCCTCTTTCTCAATGCTTTCAGAAGC ACAGTAACTGCTTACTCTGAAATGAATTCTCACCCCACTTCCACATATGCA CCCCTTGCCCACCCCTTTGGGAACACTGGCCTTAACTGCTTACCTTCAAAT GGACTCATCTGTTGGGAGATATATGCATTCTGCCGTTCAGGGGTCATTGCC ATAAGACCTGATCTCTGTTCCTCTTGCTAAACAGAAGATGAAAAAGACAA ATTAGATTACAGCTACCAATTAATAATTAGCCTTAGGATCGCTGCGTGGG GACCTAGGACTTGGCTTTGGTGCAGCAGAAAGCATGAATAAACACACCAG CATACACTCGCATGCATGCCCCACCCTCTCGAGCAAAATTCCACAGGTAT AAATAAAGTAAGATTCTGCACCTGGGTTAAAAACACAACTGCAACAGCAT AGAATGGGGCAGGAGAGACAGAACTTAATAGCAAGAGCACACAGAAAAA AGTTTTAGGCATTTTGGATGTCCATCTGCTCAGGATGGGTCAGCAGTGAG ATGCGGTCACCAAAAGAACAAATGTAACATTAGGCTGCATTAATAGAAGC AGAGTATGTAGAAGGAGGGAGGTGACAGTCCTATGCTAACTCTGCCTTGG CCAGACTATACCCACAGGAGTCTGGGCATGCCAGTCTCAGGGAGACCCAG ACAGACTGGCTGCATTCAGAGGATGGTAAGTAATGAGAGTGGGGATTGG ACTTCAAACTACCCAGACAAAGAATGGCTGAGCAAGCCAAGGATGCTGT GGCTGGGGCAGAGCAGACTGTGGGCTATGTAGTGGTGGATACCTAGCCTC TGCAGGGCTGTCATAGGGAAAGGACATTGAGAAGAGGACTGAGGCTTGTT CCTGGTGGTCCTGGC ATG A ACGGCC AGATG ATC AC ATGGTC AGGTGG AC A CAGTCTCCAACACTGGGAGTAGCCAAACACTTACTGCCAACCTCCCGCCC TTCTCCTGACTAGTTGCAGCATAGGCAATTGGGAGGAGCTTCCTGTCTCCA TCTGAAAGCTGGCTGGGTGGGCAGGGGGAGGAGCGAGCCAAGTTTCAAG GCCGCAGTTTCAGCACTCAGTCTGGGATCGGCTCAAGGAGCAAAGGGGA AGA AC ATAGCC AGGAGGGA ATAAC ATGA AGGCCCCC AGACCC AG A AAAG GCATGACTTGCTCTGAGACCCTCAGCCGGTTGGTGTCAGGTTGTGACTCG GATCCAGGTCTGACTCCCAGTCCAGTGCTTGAAGCCTCACCCCACACAGT GAGGGGAGCCCGGCCATCTCTGCTCAACTGCTGCCATCTCTCTCCCCTTCT CAACCACCAAGGCAGCTCTGTCTGGGAGCACAAGCTCCAAGTCCACTTTC TGGTCTGTGTCCCCCCCAAGATGCCAGAGGACTTGCCTCTACAACACGGG CTGCCCGTGCAGTGCCTGCTTTTCCAGCAAAGGGCTTCTGGGAACCCTTCT CTGCACTCAGTGGGGCTGGTGGGAGTGGGGCGGGGTAGCGACCCAGTGCT TGGGACTGTGCCCAGCTCTCAGGCCTGGCAGCAGTTCCTGGCCTTGGTTCC TGCCAAGGCAGAGAGGACAAACACATGGCACCGGGAAGACTACACCAGA AGCGATTCCACCAGACTGGGGTTTGCTTTTCTATCCCGCCCTTAGCCTGCT TCCTGTCCTGGTCCCTGCCTCCCCCTCCACTGGAGCTGCCGTGTGGGCAGT GAGGGGCTGTTTCTCAGCTGCCCTATGGAGCTGCCCTCTCCCTGCCAAAGC ATTGGCAAGGCGGCAAGGGGTGGGGGTGGGGATGGGGGGTGGGATCTGC CTTCTCAAGCTCTCATTATACTGAGCACGTCTCACCCATTATTTTATGTCAT CTAGCAACACCCCATGTGGACACTGAGGAGCATGGGGGTCACATGACCAC TGCCCAAGGCCACACCATCCGGATCTGCCTGAGATGGTCAGGGTTGGCAG CCATTTCTGAAGGCAGTCCTTTCGCTTTGGCTCTTCTTGTACCAGTCTCAG GACATCAGGGCAGAAGATCTACAGTCCCCAGCTTACTGATGTGACAGCAG AGGCTCAGAGAGGTTAAATGACTTGCCCAAGGTGACACGGCTAAGAAGT ACAGTATCTCCTAACTGCAGACCAGGTGCTTCTGCTGCTTCTGGGGACAG ATTCCTGCGTGGCTGGCTAGGTCTAAACGGTCCTTAACTCCATCCCCACCG GTTGCTGCATTAGTTTCATCAAATAACACAGTTGTACAGAGGTAGGGGTT CAGGGGCAGGGGCAGATGGAGGCTGGAGAGTGTGACTAAGGAAACAGCA GGGGAAGTGCGGTAAAGTCCGAAGGGAGGGACGGAAAGAGAAAGCCAA GCCCAGGGGCGTGCCAGACAAAAGGAAAGGCCACGCCGGGGCAGGGCAG GCTTCAGCGGGTGCTGGGGCGTCTTCATCCCGGGAAGCACACATTCCAGA GGACCCCGGAGTCTAATGGAAAAGCTGGCCAGCCTATCACTATGGAAACT GCCAAGGCCACACAGCGCTACGCGTATTTAAATGTGCTGGAGTGTTGAGA TACTGTAGTGGTGGGATGCTTAAGTGCTGGGGTGCTGGGTGTTGGGATGC CTAGGTGCTGGGGTGCCGAGATGCTGG AGTACTAGTGTGCTGGGATGCTG AAGTGCTGGGGTCCTGAGATGCTGCAGTGCTAGGGTGCTGAGATTCTGGG CTGCTGGAGTGTTGGGGTGCTGGGATGCTGGAGTGCTGAGATGCTTGGAC AATGGGGTGCTGGAATACTATGGTGCTGGGGTGCTGGGGTATTGAGATGC TAGGGTACTGGGATGCTGAAGTGCTGAGATCCTGGAGTGCTGGGCTGCTG GGCCACAGGCTCTTGAATCCATTCGTCTGCCCAGGGGAAGAAACCAGAAG ATAAAGAGCTAATGAAGGAGCTTTGGTTGAGAGGGAGGAAGTAATGGAA GGAGCAACATCTTGTGGAGGAGCAGGAGAGAATGGACCTCAGGTTGGGA GAGAGGGCCAGGCTACAGGCCAGAGAGGCAGAAGGATTCCAGCAGAGTG TGGGCTCCAGGAGCCAAGGGGAAACAGGTTTCTGGGAGGAGAGAGTCCA GTACTGCTGAAGTGGCAAGTCCGCTGAGGACCAGGAAGCTTCATTTGGCT TTATGACCAGGAGGAATTTGGAACTGTGACTAGAGTACTTAGGGGGAAGG AGGCAAGACTGGAGCCAGATTGCTCTGGGTTGAGGGGTGAGTGGGAGGT GAAGCAGGGCACTGTCACTCCTTTGAAGGGTGGCAGAGAGCTGGAATTGG TGCTGGATGGGCTGTGGGGTGACAGGGTCATGTGGAAAGCCCCTGGGGGG CACCTGGAAAAGGAGAAGCTGACAGTACAGTGAGAGGACAGCTAAGGGA AAGCGGAATGGCAGAACACGCACTGCCAGGAGGAATGAGGATAGGGTCA GGAGTGCCAGGGGCAGTGAGGCCAGCCTGGGGTCAGGTGGCAGGACGTG TCCAGGAAGCTGGTCTGCACTGCAGCCCACACTGGCTCAGCCTTGAGGTT CCCTGTGTGGTTGGGGTAGGAAGTTGAACCCTCTGGGAATGGAAGATGGA ACCAGCTCTGCGAGCCAAGCTCAGCTTTTATCTATGGGTCTCTGAGGGCTG GCAGAGCTGAGTGGGGACAACTGTGATCCGTGAGGCTCTCAGGTTGAGGT GGCCCCTCCGGGAGGGCTTCATTTTCCCAGCGGGTAGGTTCTAAGCAGCA GTGGCTGGGCAGGTGGGTCCAACACAGAGCCAGAGAAGGGTGAATGGGC CTCCTGGCACCCCACCCCTGCTGCCCCTGAGCTCAGTGATGGAGGGGGAC AGC AC AGCTG AGCCC A AGTGCTTTGGTGTGGCCCTG AGGG A A AGCTGC AG CCTGCCTGGGGCCTGGCATGGATGGGACACTTGAGGCAGAGGGACAATA GTGGGCGCTGCAGTGAGGCTGGCTCTTGGAGAGGTTTCCTGAGGAGTGCT GCCTGAGACGGGCAGGGAGAACAGAGACAAAGTTGGTGACAGGGAATGA AAGCTGACTGAAGGACTTTACCCAGACCTATGAGGATATCTCTCTCAGCA GGAAGC AGGAGGGGACTGTGTGAGGACTGGCC AAGAGCTGGAGTGTTGG GAAAATGACTCTTTCTCCGACCCCTCTGTCCTAGCTCTGGCCCCTGGACTG CGGAGGTCTGCTTCCACCCCCATTGGTCGATCGTTGTCCCTTGTCACAGCC ATTGAGAATTTTGGCAGGGAGCATGTTCTTAGAGCATTTTTAGGCTCTGCG GGACATAACAGCTCTGCCTCAGAGCACATGCCTTTCTCAGCTCCTGAAAG CCACTGATCAAATTGGAACATTTTGTACCTTAGGGATGAGGATATCAACT CTCCCAGCCACTTAGAGGGATAAATGTGATGATGCATTCAATTGTGACTA CATCTGATCCCAACTGTTGCTTCAGCTGCTCTCCTATAGCACATGGCGGGA GGCGTGCATCCCAGTAGCTACCTCCCCACTTTTGGGGAGATGTGGTTCCAT CCATGAAACCTGGGTACCCGCCTACCAGGTCCTGGCCTATCAGGTGGCAG GGTCTGGTCAAAGAAGGGCATGTGTGGTCTTCAGCAAGGGAGACAGGAC GGTGGTGCAGAGCGTCTAGACCCTCAGGGCAAGTCTCCCCCACACCTGCT CCCGGGGCAGTTGTCTTTGTGACCTCCCATCCCCCTCTGTTTCATCCTCTAT AAAATGAGGGGCTGAGCCCCAAAATAACAGGCTTCTTTGCCATGATGCAA AACTGCTGAATCTTTCTTTCTGACACACAAGGCATCGAGCAGCCTCTGAA AGAACCAAAGCCACTAGCAGGCTTCCTGACTTGGGTTTGTAGGTACTGAA TACTCCCTTGAAAAATAAAAACATAGAGGCACTTTTCTCCTGGCTGTTTAT TACAGAACGAAGAAAAAACACACTGGCTTGAAACAGACGCCAGATTTCA AATGTAGAGGTGAAATACGAGGTGGCAATTAAAATGTGATTACAGAAAG TCTGGACACTGAGAAAAGTTTACAGGACAGTGGGTGTGGGTTTTCTATAA CAGACACTTAAATATACATGACGATAATTGCAGATAGAAACCATCAAAGA CAAACCCCAAATCAACTAATAATGTTTACAGATGTTCCCCCCCAAACCAC AGAGCCTTACATCAAAACAAATACTGAAAGGCTTTAAACCAGGAACAGCT CGCCTTAACCCCACGAGGGTGCACACAAGCTGGGCTTTTTCTCTCGGTCTG AATGGTAAAGGGAGGAGGATACTCTAGCTCCTCCAGGTGGATTGCTGAGA CAGGGCTCGGCTCACACACTGTCTCTGCGCCTCTCCCAAATCTGGAGAAC TCTCCCAGCCTCCTGGTAAAGTGTCTCTGTGGGGCACTTAACGATAAAAC AGCTTCTGCTGTAAAGCTCATTAGGAAAGAGCTAGCGGAGACTGAAAGGT TCGCAAAAGAGATTAAGAATCACACAAGGCAATAGGATTTTTAGTGAACA TAGAAATAAATGGCCAAGTGGTTTTCTATTTGGCATTTGTCAACTTGCACA AC A ACTCTTGGTC AT ATCC AC ATTGCTC ATTGC ATTAA A ACC ATAAGCGAC TCAGCCACCTAGCTTAACAAGGTATCACTGGAGCAAACAACACGGTCTGC ATATTTGTAACATTGTATAATAAACACAAAACAATGCATAGTAAACACAA CTCTACTGAAACAAAAGCCGTCGCTTTATTTACAAAGTCACAAAATGAAG TATAAATACTTCTGTCATTAATGTTTAGGAAAACCATTTACAAAATTTTCA AATATGTACACGTAGCTTGAAAAATCACCAGCTTTCCATTTTGTCACAGGT AGAGAGAGGGATAAGCATGGGCTGACAACACCACTCAAATTGTAACGGG AGACAACTGCGGGTATGGATCGACACCACTTCCTAGAGTGATGTCACCAT GGGGGTTTCTATGGGCATCCTGCTCAGATTTAAAGTGCCCCAGCATCCTG GGTGACTTGCCCAGAATTCTGGGCTGTGGCATTTTGAGCAGCAGCATGCT GTTCCAAAATGTCGTCGATCAGCCTCAAGTTGCACACCCAGTCTTCATCTG GGCTCACACAGGAGCCTTTCAAGAGAGCTTCAATGAAATCTACCTCATTG CAGTCAGGTGACGAAATCAGATCATTTAGTGGGGGTTGGGGCTGGCGCAA AAAGTCGGCAGGTGGCAGCTCAGGGGGAATATCCGTTCTGTCGAACGGAC CTGGGAACTGGCTGGCAGCAACGGCAGAAGCAGCAGCAGCGGTGGCAGC AGCAGCCACATAGCTTGGTGGCTCGATGCCCTGTATGGGGCTCAGGGGAC TAAAGCTGGCCATACCCTGCTGGAGGAACTTGGTGGTGTTTGCTACAGGC ACCGGGCCCTGTACCGGGCTCTGCCTGAGGCTCTGGCTGCCCAGCAGGCT GAAGCTGGGGTTGTTGGCCAGGGGCACTTGTGTTCCCATCGCAGCGGGCA CTTGTGCCTCCCAATCAGATGGCCTCTGAAGGCAGGCCTGGCCAGAAGGT GAGTGCTGCTGAACGCTATTATCCACTTGGCTGAGGGGTGTTTTCCCCGAA ACTGCTGTGGTCACAGCTGCTGCCGCTGTGACCCATGCAGCATTGTTGAA CGCAGTGGGCATTCTTGGCACACTAGGCCGTCTGAGCTGGTGGGGACTCA AGGACTGGGTGCCCAGGGAGCTGGGACAGAACCCAGGCAGGGGCACTTC TGGTGGGGTGGCCTTGGGGCTCTGCATATGCTGGCAGACAGAGTCAAGTC TGCCCAGGGGAGTCTGGCCTGAGTGTGAGAGGATGGGACACTGGGGGCT GGAGGTGAAAATTCCTTGCCGCTTCCCCAGAGTTGGTGAGATCACTCCCA TGCCCGGCGCGCGCGTCGACGTGAGCAAGTGGAATGGAGTGGCAGGGAA TGAGTGAGGGAGACAACAGCTCAGTAGGACCTTGTGGGCTACTGGAAAG ACTTTGGCTTTGACTCTCAGTGAGATAAGGAGGTATTTGAAGGAGTTTTAG C AG AG A AGG A AC AATTCTGTTTATTTTT AGGAGCTCTTT AT ATATTTAGGA AATTACTTCTTTGTTAGTCATATATTTTGGAATTTGTTGTTCCCCGTTTGTC ATTTCTGTTTCTACATTTTGCTTATAATATTTGTCTATGTAAACCTTAATAT GTACTTGGTTAAATTGATAAATTCTTTGCATAATTAATAGCTAGCTATTGT TGGCCTACCTATAATTAATTTTTGCTTTATCTAGTAATAGGCCTGAATTTTC ATTTGGGATCCAGCCCTCTCCCTGACACTATGCAGGGCCTGGGAGACTAA AGCCTGCTGGAAATAACTCCAGCTTGGCCCTGCCAAACATTACAACACCT AGCACCTGCTCTCCCCACCATAGTGACTGATTTGGGCAGGAATGAGACCA CAGCCACTCCAAGAGCCCAGCTGTTGGCTACCAGTAACATCTTCTCCTCTA AACAGGAGATCCAGCTATATTGAAGGCTTCCAAAATTACCCTATGTCTAC CTGTCACAGGAATGTTCAGAGGACTAAGCCTCTGCCTTTCTCCACCCCATA CTTACTTCATATGGAACTATGAATGGGCAACTTCACAATTTATTATTTACG TCACCTAGATGCAAGTCAGAGGGAGAAGCAACAAATCAGCAGTACCTAA GAAGTTCTCAAGTCCTCAAGGTGAACTTTACCTATATATACAAAATATATT TGCCTCTTTCCTTTAACCCTTTTTCTTACTCCCACCACTTTCCACCCAATGT GTAATAATATTAGTCACTTCATAGAGTTCTTATGAGAAGTTAAATGAGGA AATCATGTAAACCACTGAGGGCTGGCAACAAGTTCTTAATAAGTGCTATT GAGTTATGAGTATTATTATCTCCCTTGGGCTGGATTCACAGTGTCACCTCT TCTGGGACCATAGTAAGCCAATGGTTCCAGCTCCTTCTATTATGTCATTTT AATTTGGGGAATAGCTTAACATATTAGATGATCCTTTACATGCCTGAGCG GTCTTTCTTTGATGGTATTTCTATGATCTTATGAAGAGCTAGATGTTGCCG CTCCATTTTATCTTGCTGGTGGCAGCTGGTGTTTAGCCAACTTGTCTTTTTT TTTCTGTCTTTATATTTTTGGTTTTGTTTTTTGGCAGAAGCCTGTGTTTTTCA GATTACTTTTTGCTTGTGTCTACTTTTTCCCAAAACAATATTACATATTAAA CTTATTGTATATTTAAGTTTATTTTATGCATCAAAAGTCGCATTGAATTTCT GTGCTATTGTGGGGC A AATTGAC ATC AATGTAATTTA ACTC ATTTTATCCC AAATTAGGCTATGTCTTTCCATATACTCAAATCTTCTTTTATGTCCCTCAAC AGTTTCCTTCATATAAATATTGCATATTTCTAATTAAGTTTATTTTTCTAAT CTCCATTTTGGATGTCTTAAATATAGTCTTTTCTCCCTTTGCTCTTTATCAA AGATCTGCAAACTATGGCCTGTTTTTGTAAATAAAGTTTTGTAATAAAGTT TAAATGGCCACTCCCATTTGTTTACATGTTGAGTATGGCTGCTTTCAGACT GAAATGGCGAATGAAGTGGTCATAACAAGACCATATGGCCTGGAAAGCA AATATATTTATTATCTGGCAATTTACAAAAAAAGTTTGCCAACCTCTGCTT TATATTTTTGATCTAGTTGTTATTTGAAAATAAAGCTATTGATATTTGTATA

ATTTCTTTTAGTTTTTCCAGGTGTAACATCTGTAAATATTAATAAATCTATT AGCTAATATTTCATTTAGCATTTTTATATCTATACTCACATGAGAAGTTTT CTTTAATTTTCTTTTGTGTTTATCAACCTGTGTTCACTCAGGAAAATGAAG CCACTTAAGTATACCAGGAGTGGAGGGTTTCATATAGAAATGAGGGCTTA TTTGAATTTTGGGAAAGCTGATGAAACAGGTCAGCAGAGTTACTGCAGAA GACCAGGAAAGTTACATCTGGAAGGTTAGGGAAGCAGACACTAAAGACT TAAGCCTGAAGCATAGAAGTAAGGATCCGTCAATACTTATTGAGAAACTT CTGGAATTCTCAAGAACCTCTGAGAAATCTCCCATTCTGTGGCAATAAAG TGGTGGTACTCAAGCCATCACCAACATGGCTCATATGTCTCATTGCACTAT GTCCAAAGACAAATGGCCTCTGCTTCTCTTCTGTCTCCTAAATCTCTTAAG ATTTCCTCTTATTTGTTTAAGTCTAACCCAGAAGACTTACATGGCAGGTAA TTCTGGGAAATGTATTCCCTTGTTTCTCCAAAGGGGTGGGGATAATACCA AGTTGACAACAGAAAATCTAGCACCCTTTTTAAGGTTTTGGCATCAGTGA TTTCTTGGTGCTCTAAATAGTTCTTATGATTATTCCATGGGTGCTTAAAAA GAATATGTATTCTCTGATTTAAGAATATGTAATTGATTTAACTATTGATTA GATATTAATAAAATAGATTTTTAAATTACCTTATTAATTTGGTTAAGTCCT CCATGACTACTTATTTTTTGACTATTTGTTATGGAAAGGTGTAGCTAAATA AATATAGAACTACTCTATATGTGTTTCTAGTTCTCTATGTATTCTCTACATT TTGCTTTATAATTGTTTTCTTTCTTAGAGACAGGGGTCTTGCTGTGTTGCCC AGGCCAGAGTGCAGTGGCATGATCATAACTCACTGTAGCCTTAAACTCCT GGGCTC AAGC A ATCCTCCTGCCTC AGCTTCTC A AGTAGATGGG ACTAC AG AAGTGCATCACCACACCCTGCTAATTTTTTTCTTATTTTTATTTTTAGTGAT GGAGTCATGCTATGATGCTCAGAGTGGTCTTGAGCTCCTGGCCTCAAGCA ATCCTCCCACCTCAGCCCCCTGAGTAGCTGGGATTATAGGTGAGAGGCAC CATGCCTGGATTTGCTTTATAAATTTTGATATGTTACTTGGTGCATAGATA TTC ATTGCTGTTAGACCTTC ATTATGTGTTGTACCCTGCCCTTCTTTGGTTC ACTTTTCCTTGAATTCAGTCTTGTCTGATATTAATTTTCAGCTGTCTGCTTT CTTCTTATTTCCATTTTTCTAATATGCTTTTGCCTACTCATTTGCTTTCAATT TTTTCTGAGTCATTTCATTCTAGATGAGTATATTACAGTCAGTACTTAATT GGGTTTTTCTTTGTCACTGAATATGAGTGTCTCTTTTCCTGTGTATTGATAG GGTAGATATGTGTAGTATGAGTGTTACAATTTTTTAAAAAACTTTGATTTC TCTCCTTTGTTTTATTTGGGGTCTCCGGTTATTTTTTAAGGTGTATAGTGTC

ATCTTCTATTTATTTAGCTAGTTTATATTGACTCTTCAATAACAGTAGTAA ATGTCCCTACTTCTAACTCCCCTTGCCCTCCACCTTTTAATTTTGTGGGGTA TACTACTGGCCTTAGTTTTTCTAGTGGTTAATTTTATACATTTAAATAGGTT TATACCTTTTAAACTTCAATGGCCACCTTTAAAAATATTTTGACCCCACCC ACTATAAAAAATAAACATGCCATCTTACACGCACAATCTCACTTTCTCTCC CCTTACATTCCCTAATTTTTACTGGGTATTTTAATATTTCTTCATCATCGTG GTTGATTACATTTACATGTATATTGCAGTGGTAGTTCCCATAATTGTTATA ACCTTTAACCATAGTTCTACATTTATTTTTGCCACCGTTTTTCCCATTCACC TTTTGGTTCAACTTTATCTTCTAGTAGTGTCTTCAAAAAAGTGTTCTTGGA AACTACAGCCCATGAAACATTATTGCATATTTGAAAATATTTATCTGTCAC CATTACACCTGAATGACAATTTAGCTAGCTCAAATTTACTGGCTCACATCA TCTTTTTTTTGGTAATTTTTTAAGTACTTCTCTACTGTCATGTAATGTTGTA TGTAGGATAGCCTAAAGCCAACTTGTGTTTTTTTATTTTGCTGTTATAGTA GTTTGATCATTTTTGCCTAGATGCCCCATAGGATTATTTTTCTTTGTCTTTA TTTCAGGAAACCTTACTAGACTATGGTTCAGTGCTGACTAGACATTTTTTT CCTTAAGAAACACCATGCCCTTTAGATCCTTAGATTCAAATCTTTTTTAAT ATCAGTAAAGTTTTTAAATTTACATATTTGGATATTTTATACTTCTCTAGTT TTTCTTCTT A A AG A AGA AAAC ATTAATTCTGTCTTTGCCTGTCTTCC ATATC TACAATTTTCTTCTAATCACTTTTTATAATTTCCATTTCATTTTGCTCAATT GCCTCAAAGCTGCTATCTATTTTTCTGTTGTCAACAATCTGTACTCTTACAT CAGCTACTATGATAATATTGTGTCACCATTTACTACTATTTACAAGCTAAC CATAATTTCTCTGATGATTTTAGTTTTCTCTTCTACTTTGTGTCCTAGATAC AGGC AGCTCAATTTTAATTTTTGTTGCTTTTCTTGATTCCTTATACCTCTGC ATTAAGCTCTTAATTCACAAATTACTTCCATTGTTAAGTTATTGAGTTCTT AAACAAATATTTGGTCATGATTTTCATCTACTCCACATCAAAATGTTCATA CTGTGTAGGCTGTTTGCCTTTTTTGTCCTGCTATTTTCCACCATTTTGTGAC ATTTTTGTATGGATCTCCTTGTTCGTTTTTTATTATTAGTCATCCTTGAGTG AGCAGAAATCTTCCTAGAGCAGGTATTTGAAAGAAGTAGTGTGGAAGAG GGGCTGGGGCTGTGTTCTGCGTTAGTAGAAAATCCCCTAATTCTTAGAAA AATCCTTTATGATTCATGGTCTTGTGGGAGAAGGTTGTGAAGCGGGTTTTG TGAATGTGCTTCATGTGCAATAGAGAGTGACTGAGAGGTGGTTTAACCTT TCTTCTTAGCTCACAAAGAATGGCTGCTACAGAGCTGCTTAACACCCACA GTTTTTCTTCTACATGCTGCACATTTACAAAGATGGAGTATCCGTGAGTTT TCTCACTCATTCCTACCTCCCCTGCCATTTTAAAGTATCCAAGGAAGTTGG GGAGATTCCCTCAGCTAAACAGCCACCCAGATCTTATTGTTCCAAACAAA AAATTATTAGATTTGCCCTCTGAGCAGGTCACCTACTTTGGAGAATTTTGT GCTCTGCCAGCTCTCCTCAAGATTTGGAAAAGCAGTTATTTTCTATTGGAG CCCCCATCACTCCAGTCCAAAGTTCACTGCATGTGACATCCCATCTTATTT GTCTGGTTCCTGATTATAGGAGTTTCCCAGTTTCAACAAAAATACTGTCTT TGTTTTTGTTTCTCATTCATTTTTCCCCAATTTTGTTTCAAAGCGAAAAGTA AAAACATTCTTACTCTAACATCTTAAAATTAATGTCTGGGAGGAACCTAA ACTGCTCATTAATATATGCCATGCTATATGTGATATGGCAGAGACTGCTAC CTGTCCACCAGAATCTATTTATTTTTCTTGGATGCACAATAAGACTACATT TCCTAGTCTCCCTTGCAATTACATGTGACCATGTCATTAAGATTTAGTGGA TGAAATTTAAGCAGAAGCAAGGTGTGTAAATTCTAGGGCTGGCCCATTAC AAACTTTAACATGGGCACCTGTGTGTTTTTCCTTTTCTGCTTAATTGGGTTG GAGATAATCACAGTGATGCTGAAAGCCATGTGCTAAAGAAGGCAGAGCC TCCTTCTGCTTGGGCTCCTGAATGACTGTGTGGAACAGAGGCCCTTGCCAC TCTTGGGCCATTCTGGATTATTACATGCACAAAGAGTAAAGTTCTTTTCTG TTAAGCCATTAGATGTATGGGTTTATTTGCTGTTACAGCCTAGTCGAACCC AAATGATGTAGTGAGGGAGGCAGGACAAGGAAGAAACCTATTCAGTATG GAGTTATCCTTAGGGACAAGTTACAAAAATTATAAGGATCAGACACCACT GCTGGTTGATCC ATTATAGACC AGA AT AAGC AGGTGGTATGATTCC AG AA GGAGATGCCATGGTTGTGTGAGAGAGAGATTAACCATTCCTATAAACAGA AGGGTCAAAGAAAGTAGCCAGTCACCAGAATGCTTATATTAAGGAGGATT GTGACTACCTGTATATAAAGTATAGGAAGAAAATGGGGCAGGGCAATTA AGATGAATTGCAGACACAACTATTTGTGCATATTAATTTGAATTTAAATTT AAACTTAATTTAAGGATAATTTAAAGGAAAATCTTCCCTTACCTATTTCAT GCTGAAGGGGTCTTTCAAGTTGTCAAACTCTCTCTATATAAATAAGACTGC ATAATCCAGACCCTCCGGAATCCAGAGATGAATTGCATGTGGTAGCTGAT GTGTGGGAAGCCCACAGAGCAGCACTTCTGGTCATTGCCATCCAGCTAGC TGGTGCTCTCAGAAGAATGAGATGAAGAGGCATGACTTTCAAGCTTCCTC TGCCATACAAGGTGAGTTTGGGTTCCAGGCTTCTTCCTAGCCCCTGTTTCT GCTTCCTCTGATTTAATACCCAGTCACTGAGTACCTACTAAACACTCGGCA TTAAACTGAAAAGAACAGGGAAGTCAAAAACCTTTTACCAATGCCTGATG GGATAAAGACATGACCCATCCCTCAAAGAGCTTGTGGTCTTCCCATGGCA ATGCCCTTCCTCCCTAGGCCAGATGGACTGTGAAGAACAGCAGTCTCAAG TGGATTTATTGGGAAGCTCATGAAGACTCAGCTCAATGGGCCATGTTTGC AGGGGCCCCTTACAAGGCTCTGCACTTGATTTTGTAATTTTGAATTATTTT TCTCAAAAAGGCCTCCCAAATTATGTAAGCTTCAGGCCTTTAAAACTTGG ATCTGTTCCCTTGAAGCCACATAATCTGATCAGAAATAATTTTCAGGGCTC ATTGGGTGCTGGAGAATTCATGGTTAGATATTTAAATAAAAATGCAGAGA ATCTATAGGACTTTCCAACTGAGACCAAGGCTTTTTTTTTCTGGTAGCTTG ACTGCCAGGAAACAGAAAGCGGGGCATCTCCACCTGCTGGCAGAAGACC AAAACTACACCATATTGCATTGACCGCAGACATCAGGCTTTGTTTCCTTTT AAGTGAATAGGTGAAATCAATCAAGCAAACTGGAATTAGGAAACAGAAG GACCACTCACAACCGTACCTCCACAGCAAAGCTAACACTTTCATTTTGAA TAACCCCTTTCCAGCCCCTGTTTACATCAGTCACATTTTTTGCATGGACAT AATAGTGTTTCAGGCAGTCCTGCTCTACAGAAGGCATTTGTTTCGCTGACC ATAAGCATCTTATGTGCTTCTACATTGTCTTTATAGCCAGCTGTCTGAAGT TCTACATCATCCGTCAGTGATGGGTCCTGAGTAAACTCAGCAGTCTCCTCT AAGTAACTTGTATATATCCCTTTTATAAAGAATGCTGCAGTGGCCACCCTT AAGTATAGAACTTTGTCTTTCCC AGGC ATTACTTACAGGAAACTGGGGAC CAAATGCCACTCCATAGCATAGCTTAGGAGAAGTGAGATTCCAAAGGACA GAGTGGGCTGGCACATATGTAGTAGAAGACTGGACAAATGAGAGTGAAT GAGCGAGTCTGTTGTGAGTGGATGACACGCAATGCAACCCCAAGGGCCG GCTCCAATTTGCACAGTCCCTAAAACATCCCAGGGATCCAGTCTTATCGC AGTCTCATCAACATTGACAATAATCATTTAGAGAATTTTCACCATGAGACT TTTGACACTCTGCTCTGGGATTTTGGAGTTCCAACTGCTTTAACAATTTAT ACAGTCACTCAAATGTAGAATCCAGTGAATTATTAATGACAATTTTGCTCC GCATATTGAAAGGGCATTCTATATTAATCAATTATAAATCATGATAAATTG CTTTCTAAAAATTATACACTTGAAACCCAGAAATGTTCAAGCCCCTCACC AGTTTACATACATCCTTAACCGTGTCTTGTTAACTGGGTGACCAGGAAAC AGAGAAAGGAGGATGTACTTAATGAATAACTGGCCAGAAGTCGGAAGAC CTAGATTTTCTTCCCATCCCTTCCATAGATTTGCTCTGTGACCCTAGACAA TTCATATGCCCCCTCTGTTACCATACTTTTAAGCTGAAGGGGTTATAGTGC ATACTTTCTGAGGTCCTTCTAACACTGGAGATCTATGAGTTCATAAGATCA CATAGAGTCTTGGACTTTTCAGAGATCACTAGGCATAAGAAAGCCCTCGA GGACATCCTCTTCCTAGATCAACCACTCCAAGCTAAGACCTTAATGGGTCT ACACTGTCATCCATTTATATAGGAGAGAGGATGGTACAAGCTAAGTGAAA GTCTGTTGTCAACTCAGGAACTAGGGTACAGTTCCAGCAATCAATCTCCA TGGAGGGTTAGCAGAGGTCTGGCCTAGCCAATTAGGGTTTGGCACTGCCA TATGCTACTAGAAAACACATGCTTTCATGCCAGGCAACAGGGTGGGAAGT GCAATGCCATTAAGGACCATACCTTAGCCTACTTAAACTAGCCTGGGATT GAGTTACTGAGCTCAAAATACAATTCAGCACACTTTCCTGCCCACTGCAA GTCAGGCCAGCCCCTGGAGAACTCCAGGGACAAGGCAAGTCTGGAGTTTG TCCCCAAGGAACTCCCAGTCTAGTGGAGGAGATGGGCCCATAGCAGAGTT AC A A ATGTCC AA AATA AG AGC AC A A ATTCCTGGGTTCTT A AAGAG AGTTC AAGCACACACACACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAT TAATTTAGATAAGGAAGGGAGGTAGGGAGAGATGGTGCCAGCAATGGCT TCCTGAGGAAATGTCACTTGTGATGGGTCTTTTAGGTGAGTTGGACATTGA GAGAAAGTAATCAAAAGGGCACAAGATAGGGTAGCTGTCAGGTAAACAA AACAAAACTGTATGC ACAAAGGTCTAGAGGCAAGAAAGC ATGGGTGTGC TTGCATAGCAATGAAGAATGTGGTTTGGCCAAAGCATAGGATTCACAGAA GGACCTCCCCAGGCCATCAGTAGTCTCTCCTTCGCAGGAGTCCACTGTGG CTGGACGAGGAACAGATCAAAACAGGTCTTGTTTGAGGCTTGGCCCAACA ACAGGGCCAGCTGTTGACCAGGGCTTCCTTCACCCTTAGCTTTCTCTCCCC CTAGTGTAGTCAGAGACTCCAAAGGCAGTCCCAGAGTGTTGTGCCAAAAC AAATGCACATCAGGCTATTGTCATCAGGAGCCCTTGTCTTGTTCTGTTTGT TTTAAGAAGACATAAGTGAAATAACCAGGTTTCCGAAGAGAGGGCAGAA TTGGGTTTGTCCCCAGGGAAAATGAGGTTTCTCCACCATGGCTGTTCTCCC CATGCAGCAGCGGCCAGGGGTCCCCAGCAGCTCTTTGCTCAGTGCCCCTG ATGCTAGGCCTGATGCTTCCTATGTGGTCAGCTCAGATGTGCAACCCTTCT CCATTTTTATTATTTGAGCAATATAATCATTATTGCTTTTGTTACCCAAAAT CTTATAATCACCCATTTAAGAGTAATGTAGGAATCATTTTTCTCTTTTATTT GATGAACTAATCTTTCTGGTTGGCTCTTGAGGTGGGAGGGGTGGGCAATG GGGACCAGCCTGCTTCATCTCACTCTTTCCTAGGCCTGCTCCTTAGGCACG TGCCCCACAAATCCCACCTCCGCAGGTTGGAAAAGGGGCCCACAGAGGAT CTCAGGTATCTGAGGAAGGGCCAGAGGCTATTTGGCCTGACATCCTGGAA CATCACCAGCCAGGAGCCATAACTCCTTTAGGCTAGCCTGCCTGCAACTG GATCAGGTATAGCAAAAACGATTGATCTCACCTTCTAATTTTCAATGAGT GGGAGTGGCTGCCTTGAGTGCTGTGTTGGGAAGGAGTCTGAGGCAGCATC TGGACCCTGTGTAAATATGCGTTTCCTGTTATTGATGAAGCTTTCAAGACA AGATTTTAGAAAGCTTTGGCTCAAGTTGTAGGTTGTGGGAGGAAGGAATT AGTAAGAGCCATCATAAACTCAACATCTATTGTGTTCCAGGAGCTGTGAC CACTCCCTTCTTTTTAGTCGGGAAGGTCAGGATCTCACTATGTCACCTAGG CTAGAGTGCAATGGTGTGATTATTGCTCATTGTAGTCTTGAATTTGAATTC CTGGGCTC A AGT A ATCCTCCTGCCTC AGCCTTCTG AGTAGGTGGG ACTACT GGTGTGTGCCACCACACTCGGCTTACTCCTCCCTTTGTATCCTGCTGCATC GTTAGTGCATGATACCCACCACCCCCTCAGAGGCCCAGCCTAGTGCTGTC TCTCAGAAAAACACCCTTTGAGTCAACAGAGACAAACTTAGGGGGTGCCT GACTCCAAAGTCCAGGCCTTCCTCCTTTGAGGAACAGCATGGCTTCCCCA GGGAATATCATTCC ATGGAGACAAGGCCTGCAGAC AGATCTCATTTTCAT CTGTTCTTTTACTAGCCCTTGGAGGATTTGCTCCAAAAACCTGAGGCACTG ATGTTTACTCCAGCTTTACTTCCCCCATACCTGGTGAACAACTCTGGCCCA GCTTCCACACACCAAGGCCCATGGCTCTAATAGAGCCAGAGAGGCAGTCT CAGCTAAGAAGAGCTGTATCCTCCAGGGACCAGCAGGAACTTCCAGAGAT ATCCTACAAGCTCCAACTGCTGGGAAAAGCAGACTGGGGTCTTCCTCAGG CTCCACCAAAGATAGCCAAGTGGCAGCAATGACATGAGCACCTCATTCCT GTCCTAGACCCTGGCAATTCCATGAGAGGGAGGCTCTGAATTCACCTCTG TTCCCAGGATGTGTGACATCAATGCACATTGTCATACCAGATACACCTGC CAAACTTCAGAACCCCTGGGAAGTGACCAGAATGCCACTGAATTCAACAC CTTAGACCAATGAACATGACCTGGGTCCTGTAGGATAGGAGGCAGCAGCA GTGTATACCCAGACCTGGTCGACTCGAATAGATCTGCTAGGCGCCAAGCT TGTCAGCCTTACCAGTAAAAAAGAAAACCTATTAAAAAAACACCACTCGA CACGGCACCAGCTCAATCAGTCACAGTGTAAAAAAGGGCCAAGTGCAGA GCGAGTATATATAGGACTAAAAAATGACGTAACGGTTAAAGTCCACAAA AAACACCCAGAAAACCGCACGCGAACCTACGCCCAGAAACGAAAGCCAA AAAACCCACAACTTCCTCAAATCGTCACTTCCGTTTTCCCACGTTACGTAA CTTCCCATTTTAAGAAAACTACAATTCCCAACACATACAAGTTACTCCGCC CTAAAACCTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAA ACTCCACCCCCTCATTATCATATTGGCTTCAATCCAAAATAAGGTATATTA TTGATGATGTTT

Gther embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.

Claims

WHAT IS CLAIMED:

1. A nucleic acid molecule comprising a low homology packaging signal cassette flanked by a recombinase recognition sequence, wherein said packaging signal cassette comprises a modified adenovirus packaging signal, provided that said modified packaging signal has low homology to a wild-type adenovirus packaging signal.

2. The nucleic acid of claim 1, wherein said recombinase recognition sequence is loxP.

3. The nucleic acid of claim 1, wherein said recombinase recognition sequence is frt.

4. The nucleic acid of any one of claims 1-3, wherein said modified packaging signal is less efficient than said wild-type packaging signal.

5. The nucleic acid of claim 4, wherein said wild-type packaging signal is human adenovirus serotype 5 packaging signal.

6. The nucleic acid of claims 5, wherein the modified packaging signal comprises at a maximum, 23 bp of contiguous sequence homology with said wild-type packaging signal.

7. The nucleic acid of claim 5, wherein said modified packaging signal is about 2-3 times less efficient than said wild-type signal.

8. The nucleic acid of claim 6, wherein said modified packaging signal comprises two to six A elements, each A element having a consensus sequence of ATTTGN8GC.

9. The nucleic acid of claim 6, wherein said nucleic acid is a plasmid.

10. The nucleic acid of claim 6, wherein said nucleic acid is a helper virus.

11. The nucleic acid of claim 10, wherein said helper virus does not contain an El gene.

12. The nucleic acid of claim 11, wherein said helper virus comprises an E3 region with an insert of about 2.9 kb.

13. The nucleic acid of claim 12, wherein said insert does not contain a promoter sequence.

14. A nucleic acid comprising an adenovirus E3 gene having an insertion of at least about 2.7 kb, provided that said insertion does not contain a promoter sequence.

15. The nucleic acid of claim 14, wherein said insertion is a human intron sequence.

16. An adenoviral helper virus for production of helper dependent vectors comprising:

(a) an adenovirus genome having an El region deletion;

(b) an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP site, a modified packaging signal and a 3' loxP site, wherein said modified packaging signal has low homology to and is less efficient than the wild-type packaging signal; and

17 . The virus of claim 16 wherein said adenovirus genome is human

Adenovirus type 5.

18. The virus of claim 17 wherein said modified packaging signal comprises at a maximum, 23 bp of contiguous sequence homology with said wild- type packaging signal.

19. The virus of claim 18, wherein said modified packaging signal is about 2-3 times less efficient than said wild-type signal.

20. The virus of claim 19, wherein said modified packaging signal comprises two to six A elements, each A element having a consensus sequence of ATTTGNgGC.

21. A vector comprising the adenovirus of claim 16.

22. A cell line expressing El and infected with the helper virus of claim 16.

23. The cell line of claim 22, wherein said cell line further expresses cre recombinase.

24. The cell line of claim 23, wherein said cell line is 293 cre cells.

25. A helper-dependent adenovirus vector comprising: a) a 5TTR; b) a packaging signal; c) at least one heterologous expression cassette; d) a human genomic stuffer DNA; e) an optional E4 non-coding segment conferring a selective advantage, wherein the E4 element is located between nucleotide -400 from the right end; and f) a 3' ITR; wherein the overall size of the vector is between about 28 kb and 36 kb, and wherein the only adenoviral sequences present are the ITRs, optional E4 non- coding segment and the packaging signal, and wherein no bacterial origin of replication or bacterial marker genes are present.

26. The helper dependent adenovirus vector of claim 25, wherein said optional E4 element is present.

27. A plasmid vector comprising a) a 5' ITR; b) a packaging signal; c) at least one heterologous expression cassette; d) a human genomic stuffer DNA; e) an optional E4 non-coding segment conferring a selective advantage, wherein the E4 element is located between nucleotide -400 from the right end; and f) a 3' ITR.

28. A helper-dependent adenovirus comprising in a 5' to 3' direction: a) a 5' ITR, b) a packaging signal cassette directly joined to the 3' of said 5' ITR, c) a first stuffer DNA at least about 1 kb, d) at least one heterologous expression cassett, e) a second stuffer DNA at least about 1 kb, f) an optionally present non-coding E4 segment at least 400 bp in length; and g) a 3' ITR, wherein said 3' ITR is directly joined to the 5' end of said non-coding E4 segment if present; provided that said helper-dependent adenoviral vector does not encode one or more proteins needed for viral generation, is about 28 kb to about 36 kb, and has a GC content between about 50% and about 60%.

29. The helper-dependent adenoviral vector of claim 28, wherein said virus is between 30 and 36 kb in length.

30. The helper-dependent adenoviral vector of claim 29, wherein said first stuffer and said second stuffer are derived from inverted mammalian non-gene or intron sequences.

31. The helper-dependent adenoviral vector of claim 30, wherein said virus does not encode for any adenovirus proteins.

32. The helper-dependent adenoviral vector of any one of claims 28- 31, wherein said optionally present non-coding E4 region is present.

33. The helper dependent virus of claim 32, wherein said GC content is between 52% to 57%.

34. A method of generating helper-dependent adenoviral gene vectors in a cell line expressing El and cre recombinase comprising: a) infecting said cell line with a helper-dependent vector comprising: a 5' ITR, a packaging signal, at least one heterologous expression cassette, human genomic stuffer DNA and a 3' ITR, wherein the overall size of the helper-dependent vector is between about 28 kb and 36 kb, and wherein no functional adenoviral coding sequences and no bacterial origin of replication or bacterial marker genes are present; b) infecting the cell line with a helper virus comprising: an adenovirus genome having an El region deletion; an excisable packaging signal cassette replacing a wild-type packaging signal, the excisable packaging signal cassette comprising a 5' loxP site, a modified packaging signal and a 3' loxP site, wherein the modified packaging signal has low homology to and is less efficient than the wild- type packaging signal; and an optional insertion element comprising at least about 2900 base pairs of non-adenoviral DNA inserted in the E3 region without deleting any part of the E3 region; and c) obtaining the generated helper-dependent viral vectors.

35. A method of generating a helper-dependent adenoviral vector comprising: a) producing a cell comprising

(i) trans functions needed for adenovirus generation; and (ii) said helper-dependent adenoviral vector, wherein said helper-dependent adenoviral vector comprises the necessary cis functions needed for adenovirus generation and at least one heterologous expression cassette, and said helper-dependent adenoviral vector does not encode for any adenovirus proteins, is about 28 kb to about 36 kb, and has a GC content between about 50% and about 60%, and b) generating said helper-dependent adenoviral vector.

36. The method of claim 35, wherein late proteins and either E2 proteins or E4 proteins, or both E2 proteins and E4 proteins are supplied by a helper virus present in said cell.

37. The method of claim 36, wherein said helper virus comprises a low homology packaging signal cassette flanked by a recombinase recognition sequence, wherein said packaging cassette signal comprises a modified adenovirus packaging signal having low homology to a wild-type adenovirus packaging signal.

38. The method of claim 37, wherein said recombinase recognition sequence is loxP and said cell expresses Cre recombinase.

39. The nucleic acid of claim 37, wherein said recombinase recognition sequence is frt and said cell expresses Flp recombinase.

40. The method of claim 37, wherein said modified packaging signal is less efficient than said wild-type signal.

41. The method of any one of claims 36-40, wherein said wild-type packaging signal is from human adenovirus serotype 5.