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WO1999006562A1 - Procede permettant de readministrer un vecteur viral adeno-associe via l'immunodepression de l'hote - Google Patents

Procede permettant de readministrer un vecteur viral adeno-associe via l'immunodepression de l'hote Download PDF

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Publication number
WO1999006562A1
WO1999006562A1 PCT/US1998/015794 US9815794W WO9906562A1 WO 1999006562 A1 WO1999006562 A1 WO 1999006562A1 US 9815794 W US9815794 W US 9815794W WO 9906562 A1 WO9906562 A1 WO 9906562A1
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Prior art keywords
antibody
patient
aav
raav
mice
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PCT/US1998/015794
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English (en)
Inventor
Varavani Dwarki
Shang-Zhen Zhou
John E. Murphy
William C. Manning
Jaime Escobedo
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Chiron Corporation
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Application filed by Chiron Corporation filed Critical Chiron Corporation
Priority to EP98938125A priority Critical patent/EP1002078A1/fr
Priority to JP2000505303A priority patent/JP2001512142A/ja
Priority to AU86721/98A priority patent/AU8672198A/en
Priority to CA002297490A priority patent/CA2297490A1/fr
Publication of WO1999006562A1 publication Critical patent/WO1999006562A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2264—Obesity-gene products, e.g. leptin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention is directed to a method for providing a somatic gene therapy.
  • the present invention is directed to a method 5 for somatic gene therapy, particularly in humans, that comprises administering an adeno-associated viral vector encoding the gene of interest and an immunosuppressant.
  • the present method is useful because it allows for expression of a gene encoded by the AAV vector without inducing a neutralizing immunoresponse. 10
  • viruses proposed for gene therapy including for example, retroviruses, adenoviruses, herpes viruses and adeno-associated viruses, express
  • a retroviral vector is an integrating RNA-based vector, which requires expression of both a wild-type reverse transcriptase and integrase to obtain ultimate expression of the recombinant gene.
  • An adenoviral vector is a
  • modifying a wild-type virus that is selectively pathogenic to a species other than its intended target For example, the envelope protein of wild-type Moloney murine leukemia virus (MoMLV), whose normal host is a mouse, has been modified to be amphotropic, and thus capable of infecting other non-mammalian species.
  • MoMLV Moloney murine leukemia virus
  • Such modifications of viral envelope proteins are extremely laborious. Accordingly, it would be desirable to provide an alternative method of gene therapy that does not require genetic alteration of the specificity of a species- specific virus.
  • Viral vectors can be either integrating (i.e., requires integration into the host cell DNA to be expressed) or non-integrating.
  • An integrating viral vector provides the prospect for long term gene expression.
  • a non- integrating vector provides for short-term gene expression. Accordingly, it is an object of the present invention to provide a method for somatic gene therapy to a patient, wherein the vector integrates into the DNA of the patient's host cell to provide long term somatic gene expression
  • AAV vectors are single-stranded linear DNA integrating vectors that are non-pathogenic and can infect both dividing and non-dividing cells.
  • the AAV genome as exemplified by AAV-2, contains two inverted terminal repeats (ITRs) at opposing ends of the virus that are 145 bp long.
  • ITRs inverted terminal repeats
  • Each repeat can form a T-shaped hairpin structure which is composed of two small palindromes flanked by a larger palindrome.
  • the AAV coding region which is between the two ITRs, is divided into three regions: rep, lip and cap.
  • the function of the rep region is to encode for four non-structural proteins that regulate of AAV DNA replication and expression.
  • the function of the cap region is to code for the three structural proteins: VP1, VP2 and VP3 of the capsid.
  • AAV preparations are stable and can be produced at high titers (> 10' 2 particles/ml). See for example, Flotte & Carter (1995) Gene Ther.2, 357-362 and Samulski, (1989) J. Virol. 63, 3822-3828. There are recent reports demonstrating long term expression of transgenes following delivery of AAV vectors into lung, liver, muscle, heart and brain.
  • AAV viruses are relatively ubiquitous and non-pathogenic, a majority of the population of animals and humans has been exposed to one or more of the seven serotypes of AAV and has developed an immune response thereto. This response would neutralize any attempted gene therapy that employed an AAV vector of the same serotype as the immunizing strain. Accordingly, it is an object of the present invention to develop a method for administering an AAV vector to a patient in need of somatic gene therapy, wherein protein expression would not be neutralized by the patients' immune system.
  • AAV adeno-associated viruses
  • the present invention is directed to a method for obtaining in vivo expression in a patient of a therapeutic agent encoded by a gene contained within an AAV vector, the patient suspected of having an immune response to AAV, the method comprising administering to the patient in need of the therapeutic agent a replication defective recombinant AAV particle (virion) having a gene encoding the therapeutic agent; and before, during or within a short period after administering the AAV vector, transiently immunosuppressing the humoral immune response of the patient to obtain expression of the therapeutic agent.
  • the therapeutic agent is a protein, polypeptide, antisense RNA or a ribozyme.
  • the therapeutic agent is a protein or a polypeptide.
  • transiently immunosuppressed or “transient immunosuppression,” as used herein, is meant that the patients' humoral (antibody producing) immune response has been reduced when compared to the patient's immune response in the absence of the immunosuppressive treatment.
  • the transient immunosuppression of the patient's humoral immune system is accomplished by administering to the patient a pharmaceutical composition comprising For other art recognized humoral immunosuppressive agent or a combination thereof.
  • the immune system of the patient is transiently immunosuppressed by administering to the patient a pharmaceutical composition comprising an antibody that is an anti-CD4, anti-CD40 (antagonistic), anti- CD40L, anti-B7-l or anti-B7-2 antibody.
  • the invention provides a method for delivering to a mammalian patient, preferably a human patient, multiple administrations of a rAAV virion encoding a therapeutic protein comprising (a) administering to the patient a therapeutically effective amount of rAAV virions encoding the therapeutic protein; (b) prior to, along with immediately after administering the rAAV virion, transiently immunosuppressing the humoral immune system of the patient to obtain expression of the therapeutic protein; and at a later date, repeating steps (a) and (b).
  • the immune system of the patient is transiently immunosuppressed both prior to and following the first administration of the rAAV virions.
  • the administering step of the present invention is performed using one of the many conventional techniques known to the art for administering a medicament.
  • these techniques include intramuscular administration, intranasal administration, intra-arterial administration and subcutaneous administration.
  • the invention is directed to a pharmaceutical composition comprising in combination an effective amount of an rAAV virion encoding a therapeutic protein, preferably a human therapeutic protein, and an effective amount of an immunosuppressant suitable for transiently immunosuppressing a mammalian patient, preferably a human, in a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is in the form of an aerosol suitable for administration by inhalation.
  • the solution may comprise about 10 6 to about 10 16 particles of rAAV virion and a humoral immunosupressant in a sterile solution of about 0.9% sodium chloride.
  • the aerosol may be contained in a sterile pneumatic aerosol generator reservoir, such that an aerosol of the solution is produced at the rate of about 8 to 12 liters per minute at about 30 to 50 psi of compressed air.
  • the pharmaceutical may be in lyophilized form, which needs reconstitution with a suitable carrier such as 0.9% saline, D50 water, Ringer's lactate and the like.
  • Figures IA and B disclose leptin expression by rAAV vectors.
  • Figure IA is a representation of the Western Blot analysis of leptin expression in vitro from supernatant harvested from human 293 cells infected with IX 10 9 AAV- leptin (lane 1); 1X10 10 AAV-leptin particles (lane 2); mock infected cells (lane 3); or cells transfected with 2 ⁇ g of pCMVKm201 -leptin (lane 4). Quantitation of leptin expression for lanes 1-3 of Figure IA as reported in ⁇ g leptin/10 6 cells/day by radioimmunoassay is shown in Figure IB.
  • Figures 2A and 2B show the effect of rAAV-leptin treatment on body weight and food intake, respectively, in ob/ob mice which received subcutaneous injections of either 10" particles of rAAV-leptin (O) or saline ( ⁇ ) and weights were monitored three times weekly. The mean ⁇ SEM of ten mice in each group was measured. For simplicity in Figures 2A and 2B, only one time point is shown for each week.
  • Figure 3 is a photograph showing the physical appearance of mice following rAAV-leptin treatment. Mice were photographed six weeks after being treated with rAAV-leptin (left) or saline vehicle (right).
  • Figure 4 shows the results of measurement of circulating leptin levels (ng/ml) at weeks 5-14 post rAAV-leptin administration in ob/ob mice (dotted bars), relative to ob/ob mice receiving saline (solid bars), and C57 mice (striped bar).
  • Figures 5A-5C show the results of measurement of the effects of rAAV-leptin on glucose metabolism and insulin secretion in treated and untreated ob/ob mice. Mice were fasted for eighteen hours and bled for determination of fasting glucose (Fig. 5A) and insulin (Fig. 5B), six weeks post-injection. The values presented are the mean _+SEM of five mice. Values are the mean +, of three mice in each group. Tests were performed on fasted mice, eight weeks post- injection.
  • Figure 6 is a bar graph showing ELISA assay results from Example 4 of erythropoietin ("Epo") expression (mIU/10 6 cells/day) by HT1080 cells infected with rAAV-Epo 5xl0 9 particles; lxlO 9 particles; 2xl0 8 particles; leptin rAAV (control) and uninfected HT1080 cells (control).
  • Figures 7A and 7B show the results of in vivo administration of rAAV-Epo virions (particles) to mice.
  • Figure 7 A is a graph showing the plasma Epo concentration in mlU/ml as a function of time (weeks) in mice administered rAAV-Epo as measured by ELISA.
  • Figure 7B is a corresponding graph showing the hematocrits of four mice administered either rAAV-Epo (squares) or saline (circles)as a function of time (weeks) relative to injection
  • Figure 8 shows the results of the ELISA assay using sera from rAAV treated mice and saline treated mice.
  • Figures 9A and 9B show the results of in vivo administration of rAAV-Epo virions (particles) to baboons.
  • Figure 9A is a graph showing the plasma Epo concentration in mlU/ml as a function of time (weeks) in baboons administered rAAV-Epo as measured by ELISA.
  • Figure 9B is a corresponding graph showing the hematocrits of two baboons administered rAAV-Epo as a function of time (weeks) relative to injection.
  • the present invention has multiple aspects.
  • the present invention is directed to a method for obtaining in vivo expression in a patient of a therapeutic agent encoded by a gene contained within an AAV vector, the patient suspected of having an immune response to AAV, the method comprising the steps of:
  • every patient is "suspected of having an immune response to AAV" because the ubiquitous nature of AAV makes it likely that most patients already have an immune response.
  • the vector and a humoral immunosuppressive agent are administered in combination to any patient in need of a therapeutic agent.
  • the method of the present invention is particularly useful when a patient in need of a therapeutic protein requires multiple or ongoing treatments over a period of years.
  • the invention allows for multiple administrations to the same patient of the same replication-defective recombinant AAV particles having a gene encoding a therapeutic protein needed by the patient, and provides for expression of the desired recombinant protein even if the patient has developed an immunity to the AAV of the vector particle.
  • the invention provides a method for delivering to a mammalian patient, preferably a human patient, multiple administrations of a rAAV virion encoding a therapeutic protein comprising the steps of: (a) administering to the patient a therapeutically effective amount of rAAV virions encoding the therapeutic protein;
  • the immune system of the patient is transiently immunosuppressed both prior to and following the first administration of the rAAV virions.
  • the therapeutic agents that are expressed within the method of the present invention include proteins, polypeptides, antisense RNA and ribozymes.
  • the therapeutic agents are proteins or polypeptides. Because AAV has a broad cell and host range, the method of the present invention is able to transform the cells of any patient to express one of the above described therapeutic agents therein. The present invention is particularly useful for those patients that require or that would benefit from a therapeutic agent on a continuous or bolus basis.
  • Example 9 To determine which arm of the host immune response was responsible for the inability to readminister rAAV vectors, readministration experiments were carried out in class I, class II and CD40 ligand deficient mice as described in Example 9. The results in Example 9 demonstrated that the humoral arm of the immune system played a key role in mounting an immune response to the AAV transfected cells and caused their destruction before the recombinant protein could be expressed. By transiently immunosuppressing the humoral arm of the immune system of the host at the time of first administration of an AAV vector, it is possible to readminister the rAAV virions as described in Example 9.
  • Agents that are used to achieve transient humoral immunosuppression of the patient's immune system include anti-B7-l or B7-2 antibodies, anti-CD40 (antagonistic) antibodies or CD40 ligand antibodies or a combination thereof. Methods for making and using many of these antibodies, and antigen binding fragments thereof, are disclosed in U.S. Patent Application Serial No. 08/015,147 (now allowed), U.S. Patent 5,397,703, U.S. Patent No. 5,677,165, U.S. Patent 5,747,034, U.S. Serial No. 08/469,015, U.S. Serial No. 08/463,893, and U.S. Serial No. 08/606,293, all of which are expressly incorporated herein by reference.
  • agents which are useful for transiently immunosuppressing the humoral immune response in a patient, include cyclophosphamide and deoxyspergualin. See, Smith, Gene Therapy 3 (1996) 496-502. Still other agents which are useful to transiently immunosuppress the humoral immune response in a patient include anti-CD3 (OKT3) antibodies, CTLA4Ig (Bristol Myers) and anti- CD4 antibodies and FK506.
  • the humoral immune response of a patient is transiently immunosuppressed by administering to the patient a pharmaceutical composition comprising an antibody that is an anti-CD4, anti- CD40 (antagonistic), anti-CD40L, anti-B7-l or anti-B7-2 antibody, or a combination thereof.
  • the replication-defective AAV vectors and virions utilized in the method and pharmaceutical compositions of the present invention are prepared using conventional methods of virology, molecular biology, microbiology and recombinant DNA techniques. Such techniques are well known and explained fully in the literature, including, for example, in Sambrook, Molecular Cloning: A Laboratory Manual (Current Ed.); DNA Cloning: A Practical Approach (D. Glover, ed.); Oligonucleotide Synthesis (Current Ed., N. Gait, ed.); Nucleic Acid Hybridization (Current Ed., B. Hames and S. Higgins, eds.); Transcription and Translation (Current Ed., B. Hames and S.
  • Gene transfer, gene therapy or gene delivery refer to methods, techniques or systems for reliably inserting into a host cell a heterologous or a foreign DNA or a DNA not normally expressed.
  • the resultant insertion can be by integration of transferred genetic material into the host cell genomic DNA, by extrachromosomal replication and expression of transferred replicons or in a non- integrated manner.
  • Vector means any genetic element that is capable of replication when associated with the proper control elements and that can transfer DNA or RNA sequences between cells. Examples include plasmids, phages, transposons, cosmids, chromosomes, viruses, and virions and include cloning and expression vehicles and viral vectors.
  • the AAV vectors and replication-defective AAV virions utilized herein comprise a DNA encoding a therapeutic protein operably positioned between a pair of adeno-associated virus inverted terminal repeats ("AAV ITRs").
  • AAV ITRs are art-recognized regions found at each end of the AAV genome that function together in cis as recognition signals for DNA replication and for packaging the AAV vector into an AAV coat.
  • the nucleotide sequences of the AAV ITR regions for the various AAV serotypes i.e., AAV-1 to AAV-7 are known in the art and vary in size with the serotpe. Typically, the AAV ITRs range in size from about 125-145 bp.
  • the AAV ITRs of Applicants' recombinant replication-defective retrovirion need not be identical to the nucleotide sequence of the native, i.e., wild-type, sequence, but may be altered by insertion, deletion or substitution of nucleotides.
  • the two AAV ITRs may be derived from any of the AAV serotypes, for example AAV-1, AAV-2, AAV-3, AAV-4, AAV-5 and AAV-7, and need not be identical to or derived from the same serotype, so long as they permit integration of the heterologous sequence of interest into the recipient cell genome when AAV rep gene products are present in the cell.
  • the AAV rep coding region is the art-recognized region of the AAV genome that encodes the proteins required for replication of the viral genome and for insertion of the viral genome into a host genome during latent infection.
  • the rep coding region includes at least the four genes encoding the two long forms of rep (rep 78 and rep 68) and the two short forms of rep (rep 52 and rep 40).
  • the rep coding region may be derived from any AAV serotype or from a functional homologue such as the human herpes virus 6 rep gene.
  • the region need not include all of the native sequence, but may be altered by insertion, deletion or substitution of nucleotides, so long as the sequence that is present provides for sufficient integration when expressed in a suitable recipient cell.
  • the AAV vector and virions utilized in the present invention lack one or more of the rep proteins so as to render it replication- defective. More preferably, the AAV vector of the present invention lacks all four of the rep proteins.
  • the AAV cap coding region is the art-recognized region of the AAV genome that encodes the capsid or coat proteins, VPl, VP2 and VP3, that package the viral genome. For more details, see, for example, Muzyczka, Current Topics in Microbiol. 158 (1992) 97-129 and Kotin, Human Gene Therapy 5 (1994) 793-801.
  • the cap coding region may be derived from any AAV serotype or from a functional homologue.
  • the cap coding region may be altered by insertion, deletion or substitution of nucleotides, so long as the sequence present provide for sufficient packaging when expressed in a suitable recipient cell.
  • cap coding region is preferably not included in the AAV vectors and the replication-defective AAV virions employed in the present invention, it needs to be included in a helper vector that is expressed in a packaging cell that recognizes and packages the ITRs and the gene(s) positioned therebetween.
  • AAV vector means a vector derived from an adeno-associated virus serotype that includes at least those sequences required in cis for replication and packaging, for example, a pair of functional ITRs flanking a heterologous (i.e., non-AAV) nucleotide sequence.
  • any AAV vector of any serotype can be employed in the method of this invention.
  • vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, PCT Patent Publication WO 93/09239 or simply a pair of AAV-7 ITRs having one or more genes operatively positioned therebetween.
  • the AAV ITRs employed in the vectors and virions of the present invention may be the native (wild-type) AAV ITRs or they may be modified. If the ITRs are modified, they are preferably modified at their D-sequences.
  • the native D-sequences of the AAV ITRs are sequences of twenty consecutive nucleotides in each AAV ITR (i.e., there is one sequence at each end) which are not involved in HP formation.
  • the D-sequences of the ITRs are modified by the substitution of nucleotides, such that 5-18 native nucleotides, preferably 10-18 native nucleotides, most preferably 10 native nucleotides, are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native, i.e., exogenous, nucleotides.
  • One preferred sequence of five native nucleotides that are retained is 5' CTCCA 3'.
  • the exogenous or non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence at the same position.
  • appropriate replacement nucleotides for native D-sequence nucleotide C are A, T and G
  • appropriate replacement nucleotides for native D-sequence nucleotide A are T, G and C.
  • the construction of four such AAV vectors is disclosed in United States Serial No. 08/921,467, filed September 2, 1997.
  • Other employable exemplary vectors are pWP-19 and pWN-1, both of which are disclosed in Nahreini, Gene 124 (1993) 257-62.
  • Another example of such an AAV vector is psub201 as disclosed in Samulski, J. Virol. 61 (1987) 3096.
  • AAV vectors are the Double-D ITR vector. Methods for making the double-D ITR vectors are disclosed in U.S. Patent No. 5,478,745. Still other suitable AAV vectors are those disclosed in U.S. Patent No. 4,797,368 (Carter) and U.S. Patent No. 5,139,941 (Muzyczka), U.S. Patent No. 5,474,935 (Chartejee) and PCT Patent Publication WO 94/28157 (Kotin).
  • an AAV vector employable in the methods of this invention is SSV9AFABTKneo, which contains the ⁇ -fetoprotein (AFP) enhancer and albumin promoter and directs expression of the herpes simplex thymidine kinase (TK) gene predominantly in the liver. Its structure and method for making are disclosed in Su, Human Gene Therapy 7 (1996) 463-70).
  • the replication-defective AAV vectors are packaged into empty AAV capsids to produce the replication-defective AAV virions helper viruses employed in the methods of the present invention.
  • To package the replication- defective AAV vectors which are typically one or more genes positioned between a pair of ITRs, one employs a helper construct or helper virus that has AAV- derived coding sequences that function in trans to enable AAV replication, and that include the AAV rep and cap sequences.
  • the helper virus has AAV coding sequences but lacks the AAV ITRs and thus are not packaged in the capsids that are produced. This helper virus then provides for transient expression of the AAV rep and cap genes missing from the AAV vector.
  • AAV helper constructs see, for example, Samulski, J. Virol. 63 (1989) 3822-28; McCarty, J. Virol 65 (1991) 2936-45 and U.S. Patent No. 5,139,941.
  • One such AAV helper construct comprises pKS rep leap, which contains the genes encoding the AAV-2 rep and cap polypeptide sequences.
  • Additional examples of helper viruses, constructs and functions that can be employed include the plasmids pAAV/Ad and pIM29+45 (see Samulski, J. Virol. 63 (1989) 3822-28 and McCarthy, J. Virol 65 (1991) 2936-45) and those disclosed in U.S. Patent No. 5,622,856.
  • Accessory functions and accessory function vectors are non-AAV derived functions and vectors containing sequences encoding such functions upon which AAV is dependent for its replication.
  • Such accessory functions can be derived or obtained from any of the known helper viruses, such as adenovirus, herpesvirus (except herpes simplex virus type-1) and vaccinia virus and include moieties and/or sequences involved in activation of gene transcription, DNA replication, synthesis of cap expression products and capsid assembly. See, for example, Carter, "Adeno-Associated Virus Helper Functions" in CRC handbook of Parvoviruses, Vol. I (1990) (P. Tijssen, ed.); Muzyczka, Current Topics in Microbiol.
  • the heterologous nucleotide sequence(s) that are inserted into the replication-defective AAV vectors and virions of the present invention encode one or more therapeutic agents that include a therapeutic protein, polypeptide, antisense RNA or a ribozyme, or a combination thereof.
  • the vectors or virions contain from one to two therapeutic agents that are native or non-native to the recipient cell but which have a desired biological or therapeutic effect.
  • heterologous nucleotide sequences that are introduced into the replication-defective AAV vectors and virions of the present invention include a gene that encodes a therapeutic protein or polypeptide, preferably a human protein or polypeptide.
  • therapeutic proteins and polypeptides that would be suitable for expression in the methods of the present invention include the LDL receptor, Factor VIII, Factor IX, phenylalanine hydroxylase, ornithine transcarbamylase, or ⁇ l-antitrypsin; a cytokine, such as interleukin (IL)-l, IL-2 IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IL-13, IL-14 and IL-15, ⁇ -interferon, ⁇ -interferon, the ⁇ -interferons, tumor necrosis factor CD3, ICAM-1, LFA-1, or LFA-3, a chemokine including RANTES l ⁇ , or MlP-l ⁇ (see Cocci, Science 70 (1996) 1811-15); a colony stimulating factor, such as G-CSF, GM-CSF and M-CSF; growth factors such as IGF-1 and IGF
  • nucleotide coding sequences for these proteins and polypeptides are already known in the art. Even more sequences expressible in the methods and compositions of the invention include Protein S and Gas6, thrombin, Coagulation Factor Xa, acidic fibroblast growth factor (FGF-1), basic FGF (FGF-2), keratinocyte growth factor (KGF), TGF, platelet derived growth factor (PDGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and HGF activators, PSA, nerve cell growth factor (NCGF), glial cell derived nerve growth factor (GDNF), vascular endothelial growth factor (VEGF), Arg-vasopressin, thyroid hormones asoxymethane, triodothyronine, LIF, amphiregulin, soluble thrombomodulin, stem cell factor, osteogenic protein 1, the bone morphogenic proteins, MFG, MGSA, heregulins and melanotropin.
  • FGF-1 acidic fibroblast growth factor
  • Preferred proteins include but are not limited to erythropoietin, thrombopoietin (G-CSF), Factor VIII, Factor IX, Factor Xa, human growth hormone, leptin and IL-2, the DNA sequences of which are all known in the art, particularly the human DNA sequences.
  • Epo erythropoietin
  • leptin leptin from rAAV-Epo and rAAV-leptin, respectively, using the methods of the present invention are disclosed in the examples herein.
  • An antisense sequence that is expressible by the replication- defective AAV vectors and virions of the present invention is an RNA sequence that can prevent or limit the expression of over-produced, defective, or otherwise undesirable molecules by being sufficiently complementary in sequence to the target sequence that binds to the target sequence.
  • the target sequence can be part of the mRNA that encodes a protein, and the antisense RNA would bind to the mRNA and prevent translation.
  • the target sequence can be part of a gene that is essential for transcription, and the antisense RNA would bind to the gene segment and prevent or limit transcription.
  • group C adenoviruses Ad2 and Ad5 have a 19 kilo Dalton glycoprotein (gpl9) encoded in the E3 region of the virus that binds to class 1 MHC molecules in the endoplasmic reticulum of cells and prevents terminal glycosylation and translation of the molecule to the cell surface.
  • the liver cells Prior to liver transplantation, the liver cells may be infected with gpl9-encoding AAV vectors or virions which upon expression of the gpl9 inhibit the surface expression of class 1 MHC transplantation antigens.
  • These donor cells may be transplanted with low risk of graft rejection and may require a minimal immunosuppressive regimen for the patient. It may also permit a donor-recipient state to exist with fewer complications.
  • Ribozymes that are expressed by the replication-defective AAV vectors and virions in the method of the present invention are useful in treating various diseases and conditions. Ribozymes are RNA polynucleotides capable of catalyzing RNA cleavage at a specific sequence and hence useful for attacking particular mRNA molecules. In chronic myelogenous leukemia for example, the "Philadelphia chromosomal translocation" causes expression of a bcr-abl fusion protein and abnormal function of the abl oncoprotein.
  • a ribozyme specific for either of the two bcr-abl fusion mRNA splice junctions can inhibit expression of the oncoprotein.
  • exemplary ribozymes include ribozymes to hepatitis A, hepatitis B and hepatitis C. See Christoffersen and Marr, J. Med Chem 38 (1995) 2023-37 and Baarpolome, J. Hepatol 22 (1995) 57- 64. See U.S. provisional Patent Application Serial No. 60/025,616 filed September 06, 1996, and herein incorporated by reference.
  • the protein or polypeptide encoded by the genes inserted into the replication-defective AAV vectors and virions of the present invention provide one or more antigens from pathogenic agents that may be used to immunize the patient.
  • the rAAV vectors or virions are administered in accordance with the methods of the present invention, but are employed as vaccines as described in U.S. Patent Application Serial No. 09/096,966, filed June 12, 1998 and herein incorporated by reference.
  • Preferred antigens are HCV antigens, such as HCV NS3, NS4, El, E2 and/or E2a. Also preferred are H.
  • Pylori antigens VacA cytotoxin
  • heat shock protein CagA (cai antigen)
  • urease B urease B.
  • antigens useful in this invention include HSV (herpes simplex virus), gD, gB and other glycoproteins, HIV gp 120, p24 and other proteins, CMV (cytomegalovirus) gB or gH glycoproteins, hepatitis D virus (HDV) delta antigen, hepatitis A virus (HAV) antigens, EBV (Epstein Barr vims), MMR and VZV (Varicella Zoster virus) antigens, influenza antigens, rabies antigens and bacterial antigens from Bordetella pertussia, Neiserria meningitides (A, B, C, Y 135).
  • the nucleotide sequences encoding these antigens or antigenically active fragments thereof are well known to those of ordinary skill in the art.
  • active fragment a polypeptide containing less than a full-length sequence that retains sufficient biological activity to be used in the methods of the invention.
  • analog as used herein, is meant a truncated form, splice variant, mutein with amino acid substitutions, deletions or additions, an allele, or derivative of the mature protein or polypeptide which possess one or more of the native bioactivities of the full length protein or polypeptide.
  • polypeptides that are identical or contain at least 60% , preferably 70% , more preferably 80% and most preferably 90% amino acid sequence homology to the amino acid sequence of the mature protein wherever derived, from human or non-human sources are included within this definition.
  • the replication-defective AAV vectors and virions employed in the present methods and composition may also include control sequences, such as promoters and polyadenylation sites, selectable markers, reporter genes, enhancers and other control elements permitting for transcription induction and/or selection.
  • control sequences such as promoters and polyadenylation sites, selectable markers, reporter genes, enhancers and other control elements permitting for transcription induction and/or selection. The insertion of these sequences and sites is performed using conventional techniques that are well known in the art.
  • the AAV helper construct is used to complement AAV functions missing from the AAV vector which are necessary for the production of AAV virions, in particular, the rep and cap functions.
  • Suitable helper constructs having complementing functions are well known in the art.
  • the AAV vector, helper construct and adenoplasmid accessory (helper) construct are introduced into the host cell either simultaneously or sequentially, using any of the well known, art recognized transfection techniques, for example by calcium phosphate coprecipitation. Culture conditions include incubation in the range of 33° to 39°C, preferably 37°C for approximately 48 to 120 hours. The cells are collected and lysate produced using three freeze/thaw cycles by sonication.
  • the lysates are then centrifuged to remove cell debris and the rAAV virions purified by cesium chloride equilibrium gradient centrifugation. Any residual adenoviral particles can be inactivated by heating the purified rAAV preparation to at least 56°C for 20-30 minutes.
  • the rAAV virions can be purified by sulfonated cellulose column chromatography following the protocol described in Tamaose, Human Gene Therapy, 7 (1996) 507-13.
  • the AAV virions of the invention are employed in pharmaceutical compositions for the treatment of diseases and/or conditions in which systemic administration of a therapeutic substance, for example, a secretory protein is desired or for preventing infections by the organism whose antigen is incorporated into the AAV vector.
  • compositions comprising an effective amount of the AAV virions of the invention in admixture with a pharmaceutically acceptable carrier, for example, a sterile, non-immunogenic solution (such as 0.9% NaCI, D50 water, Ringer's lactate, or phosphate buffered saline) or with an adjuvant/antigen presentation system (such as alum).
  • a pharmaceutically acceptable carrier for example, a sterile, non-immunogenic solution (such as 0.9% NaCI, D50 water, Ringer's lactate, or phosphate buffered saline) or with an adjuvant/antigen presentation system (such as alum).
  • adjuvant/antigen presentation systems for example, MF59 (Chiron Corp.), QS-21 (Cambridge Biotech Corp.), 3-DMPL (3-Deacyl-Monophosphoryl Lipid A) (Ribilmmnochem Research Inc.), clinical grade incomplete Freund's adjuvant (IFA), fusogenic liposomes, water soluble polymers or Iscoms (Immune stimulating complexes) may also be used.
  • a mucosal adjuvant for preparation of intra-nasal formulations as described in PCT Patent Publication WO95/17211, published June 29, 1995 (Biocine Application Number PCT/IB95/00013) is preferably employed.
  • the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a replication-defective AAV virion having a gene encoding a therapeutic agent, preferably a protein, in combination with an effective amount of a humoral immune suppressant, and in a pharmaceutically acceptable carrier.
  • compositions that carry the vectors and virions of the present methods are administered using conventional techniques known to the art for administering any medicament.
  • the pharmaceutical compositions employed in the present invention are administered intranasally, intramuscularly, subcutaneously, intravenously or intraarterially. Formulations and modes of administration are discussed in greater detail below.
  • the nasal cavity offers an important route of administration for the recombinant AAV virions of the present invention.
  • the human nasal cavities have a total surface area of approximately 150 cm 2 and are covered by a highly vascular mucosal layer.
  • a respiratory epithelium comprised of columnar cells, goblet cells and ciliary cuboidal cells line most of the nasal cavity. See Chien, Crit Rev. in Therap Drug Car sys 4 (1987) 67-194.
  • the subepithelia contains a dense vascular network and the venous blood from he nose passes directly into the systemic circulation, avoiding first-pass metabolism in the liver. By avoiding first-pass metabolism, delivery to the upper region of the nasal cavity may result in slower clearance and increased bioavailability.
  • cilia in this area is an important factor in the increased effectiveness of nasal sprays as compared to drops.
  • viscosity-building agents such as methycellulose can change the pattern of deposition and clearance of intra nasal applications.
  • bioadhesives can be used as a means to prolong residence time in the nasal cavity.
  • Various formulations comprising sprays, drops and powders, with or without the addition of absorptive enhancers, have been investigated. See, for example, Wearley, Crit Rev in Therap Drug Car Sys 8 (1991) 331-94. It is advantageous to administer rAAV via the intra-nasal route.
  • Intra-nasal administration is easy and convenient, economical, safe (an overdose is, in most instances, treatable) and does not require medical personnel, the nasal route has been shown to be effective for the administration of a number of molecules due to the extensive network of capillaries located under the nasal mucosa. This facilitates effective systemic absorption and when the drug is administered with absorption promoters, absorption occurs rapidly with high bioavailability (see Gizurarwon, Acta Pharm 2 (1990) 105). However, when adenoviral vectors are administered intra-nasally, cellular, humoral and mucosal CTL responses result. Additionally, it is also advantageous to be able to readminister rAAV via the intra-nasal route.
  • Exemplary formulations of the AAV vector containing lactose, trehalose or mannitol for intramuscular or subcutaneous administration can be prepared by combining one part 2x formulation buffer to one part purified rAAV
  • the lactose buffer and the trehalose buffer each contains 25 mM trimethamine 70 mM NaCI, 2 mg/ml arginine, 10 mg/ml human serum albumin (HSA) and either 100 mg/ml lactose or 100 mg/ml trehalose in a final volume of 100 mis at a pH of 7.4.
  • the mannitol buffer contains 25 mM tromethamine, 35 mM NaCI, 2 mg/ml arginine, 10 mg/ml HSA and 80 mg/ml mannitol in a final volume of 100 ml at a pH of 7.4.
  • the dosage regimen will be determined by the attending physician considering various factors known to modify the action of drugs such as physician condition, body weight, sex, diet, severity of the condition, time of administration and other clinical factors.
  • Exemplary dosage ranges comprise between 10 3 to 10 14 particles, preferably 10 6 to 10 16 particles, more preferably 10 s to 10 14 particles, most preferably 10 10 to 10 12 particles.
  • nebulizer can deliver a predetermined volume of the formulation t the nasal cavity.
  • Ultravent which is available from Mallinckrodt.
  • the desired formulation of rAAV virion is placed in the reservoir of the Ultravent pneumatic aerosol generator.
  • the generator is driven with compressed air at about 30-50 psi, preferably 40 psi, generating 10 liters/min (at 40 psi) of aerosol.
  • this system is closed and all gas is inspired or expired through a filter.
  • the muscle tissue Prior to intramuscularly administering the replication defective rAAV virions, the muscle tissue may be injected with a cell proliferating agent.
  • the cell proliferating agent is bupivacaine.
  • the bupivacaine may be injected up to about twenty-four hours prior to injection of the rAAV virions.
  • About 50 ⁇ l to about 2 ml of 0.5 % bupivacaine-HCl and 0.1 % methylparaben in isotonic NaCI may be administered to the site where the rAAV virions is to be administered.
  • the cell proliferation agent may be included in the formulation with the rAAV virions.
  • 50 ⁇ l to about 1500 ⁇ l, more preferably about 1 ml of the agent may be included.
  • a pharmaceutical composition comprising a therapeutically effective amount of the replication-defective AAV virions of the invention, alone or in combination with a humoral immunosuppressant, and in a pharmaceutically acceptable liquid carrier (e.g. , 0.9% sterile saline) is taken up in a sterile syringe with a 22 gauge needle and injected under the skin on the forearm of the patient in need of treatment.
  • a pharmaceutically acceptable liquid carrier e.g. 0.9% sterile saline
  • this administration is followed up with the administration of a one or more doses of a humoral immunosuppressant, using the manufacturers recommendations as a guideline with due consideration for the age, health, sex, and size of the patient.
  • the same liquid pharmaceutical compositions as described above are used to administer a dose of the replication-defective AAV vectors or virions to a patient via an artery.
  • the intraarterial administration is performed using standard techniques that are known to the art, including the use catheters, which can be threaded through an artery to deposit the dose at a preferred tissue site.
  • catheterization techniques are employed extensively in cardiac visualization and are readily available to those skilled in the art.
  • a specific example of administering the replication-defective AAV virions to a renal artery of a patient is disclosed in Example 10 herein.
  • Example 1 details the construction of the rAAV-leptin construct.
  • Example 2 describes the preparation and titering of rAAV-leptin particles.
  • Example 3 details the in vitro analysis of rAAV-leptin and Example 4 discloses the in vivo administration of the construct in mice.
  • adenoviral gene delivery results in rapid onset of protein expression, which is extinguished within two weeks, presumably by immune response to adenoviral proteins. See Muzzin, Proc. Natl. Acad. Sci. USA 93, 14804-14808.
  • Epo monkey erythropoietin
  • the invention is further exemplified by the administration of the heterologous sequence encoding monkey erythropoietin (Epo), in mammals.
  • Epo which is produced in the kidney of mammalian adults is a key hormone involved in regulation of erythrocyte differentiation and the maintenance of a physiological level of circulating erythrocytes (red blood cells).
  • Clinically, Epo is the treatment of choice for anemia associated with chronic renal failure or for the treatment of thalassemia.
  • the biological effect of Epo gene expression is monitored by determining hematocit levels and the circulating concentration of the hormone is measured standard immunoassay or by ELISA.
  • pKm201CMV is an AAV cloning vector in which an expression cassette, consisting of a CMV immediate early enhancer, promoter and intron and a bovine growth hormone (BGH) polyadenylation site, is flanked by inverted terminal repeat (ITR) sequences from AAV-2.
  • pKm201CMV was derived from pKm201, a modified AAV vector plasmid in which the ampicillin resistance gene of pEMBL-AAV-ITR (see Srivastava, (1989) Proc. Natl. Acad. Sci. USA 86, 8078-8082) has been replaced with the gene for kanamycin resistance.
  • the expression cassette from pCMVlink a derivative of pCMV ⁇ c (see Chapman, (1991) Nucleic Acids Res. 19, 193-198) in which the GBH poly A site has been substituted for the SV40 terminator, was inserted between the ITRs of pKm201 to generate pKm201 CMV.
  • AAV leptin expression vector pc ⁇ CMVAAV-m-leptin a 511bp fragment, encoding murine leptin cDNA (see Giese, (1996) Molecular Medicine 2, 50-58) was cloned into the Xba I- Bam HI sites of pKm201 CMV.
  • the AAV vector contains a post-transcriptional regulatory element (PRE) from hepatitis B virus.
  • the PRE which increases efficiency of mRNA transport (see Huang, Mol. Cell. Bio. 15, 3864-3869), was included to increase the size of the vector genome for more efficient packaging.
  • a 579 bp fragment, from the post-transcriptional regulatory element (PRE) region of Hepatitis-B (HBV) was amplified using the primer set:
  • the resulting fragment was digested with Ascl and Mlul and inserted into an MM site between the leptin coding region and the GH poly A site. Inclusion of the coding region for mouse leptin into this construct results in a 3.4 kb packageable vector genome.
  • This vector plasmid was packaged using standard methods, and a purified stock of 1.25xl0 12 particles/ml was obtained.
  • AAV helper plasmid pKSrep/cap (encoding rep and cap protein) was constructed by cloning the AAV-2 genome, without the ITRs (AAV-2 nucleotides 192 through 4493) into pBluescript II KS+ (Stratagene, La Jolla, CA).
  • rAAV vectors were produced by a modified transient plasmid transfection protocol. See Zhou, (1994) J. Exp. Med. 179, 1867-1875. Briefly, human embryonic kidney 293 cells, grown to 60% confluence in a 15 cm dish, were co-transfected with 12.5 ⁇ g of helper plasmid pKS rep/ cap and 12.5 ⁇ g of vector plasmid pCMVAAV-m-leptin or pCMVAAV-lacZ using the calcium phosphate co-precipitation method. After 8 hr, transfection medium was replaced with IMDM + 10% (fetal bovine serum) FBS containing adenovirus type 5 dl312 at a multiplicity of infection (MOI) of 2.
  • MOI multiplicity of infection
  • the cells were harvested in HEPES buffer (2.5 ml per dish) and lysed by three cycles of freezing and thawing. The cell lysage was centrifuged at 12,000x g for 20 min to remove cell debris. The packaged rAAV virus was purified through two rounds of cesium chloride equilibrium density gradients to remove any contaminating proteins and heated at 56°C for 45 min to inactivate residual adenovirus particles.
  • the vector stock was treated with DNAse I, and encapsidated DNA was extracted with phenol-chloroform, precipitated with ethanol. Released DNA was compared to a known standard by dot blot analysis.
  • 10 9 or 10 10 rAAV-leptin particles were diluted in 2 ml of IMDM + 10% FBS and added to 1 x 10 6 human embryonic kidney cells (293 cells) plated at 50% confluence on a 6-well dish. Virus was left on cells for 24 hr. Cells were washed and 2 ml of fresh IMDM + 10% FBS was added. Supernatant was collected for Western blot or RIA analysis (24-48 hour postOV-infection). See Figs. IA and IB.
  • supernatant was harvested from cells transfected with 2 ⁇ g of the pCMVAAV-m-leptin packaging plasmid.
  • 2 ⁇ g of pCMV-AAV-m-leptin plasmid was incubated with lO ⁇ l of transfection reagent LT1 (Panvera Inc. , Madison WI) and added to 5xl0 5 human embryonic kidney cells (293 cells) seeded on six well dishes. Complexes were incubated with cells for four hours and refed with 2 ml of media. Cell supernatant was collected 48 hr post-transfection and analyzed by Western blot or RIA.
  • the level of leptin expression was quantitated using a sensitive RIA. While mock infected cells released no detectable leptin into media, cells infected with 10 9 and 10 10 particles released 47 and 290 ng of leptin per 24 hr/10 6 cells, respectively. See Fig. IB. Interestingly, we found that the packaging capability of the rAAV vector is sensitive to the size of the vector genome packaged. Inclusion of PRE sequence to increase the size of the vector from 2840 to 3430 bp helped in improving the functional titer (data not shown). This result is consistent with the findings of Dong et al. (1996) Human Gene Ther. 7, 2101- 2112, who have demonstrated a direct correlation between genome size and titer of recombinant AAV vectors.
  • mice Twenty four to six week old female C57BL/6J-ob/ob mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The weights of ten rAAV-leptin treated mice were compared with ten mice treated with 0.9% saline vehicle on a weekly basis. Following anesthesia with a mixture of ketamine and xylazine, 50 ⁇ l of normal saline or normal sale orlxlO 11 rAAV particles was injected into the tibialis anterior (TA) muscle. In some experiments, both legs were injected on two successive days, while in other experiments twice as many particles were injected on one day.
  • TA tibialis anterior
  • rAAV-leptin treated mice gained significantly less weight starting from week 1 until the end of the observation period. Treated animals began losing weight by week 4 and continued to lose weight until week 8 at which time weight began to stabilize. At week 8, the average weight of these animals is much closer to the age matched C57 control mice than to untreated ob/ob mice.
  • Monitoring of food intake was begun in the third week following injection. Pre- weighed standard mouse food was added to cages (containing five mice) each evening and the amount of food consumed was measured the following day. The reduction in food intake of treated mice corresponded with the extent of weight loss.
  • mice As shown in Figure 2B, at the earliest time points monitored (day 18-23) rAAV leptin treated mice ate a daily average of 3.4g of food per mouse as compared to an average of 5.1 g for saline treated controls. The following week (day 24-29) the mice ate an average 2.9 g of food per day and untreated mice at 4.6 g/day. From week 4 through week 7, the leptin treated mice consistently consumed an average 1.9 g of food/day and by week 9 the consumption was —2.3 g/day. During weeks 10 and 11 consumption, in the leptin treated mice, plateaued at 2.75g/mouse/day.
  • the level of circulating leptin was measured at 5, 7, 9 and 11 weeks after intramuscular delivery of rAAV-leptin.
  • Blood was collected from isofluorane anesthetized mice by retroorbital bleeds and separated into serum, the levels of circulating leptin were measured using a Lincomouse Leptin RIA Kit (Linco, St. Charles, MO).
  • the serum leptin levels from five AAV- leptin treated mice and five saline-treated controls were measured.
  • Serum was collected from mice at the indicated times and leptin levels were measured using the Linco Ria kit. Values are the average of 5 mice _+ SEM. Week 7 mice were fasted for eighteen hours prior to serum collection.
  • mice tested at weeks 5, 9 and 11 were fed ad libitum prior to serum collection. Serum was not collected from C57 mice at week 5. As shown in Figure 4, at week 5 average leptin concentration for the treated mice was 1.7 ng/ml, with a range of 1.3 - 2.34 ng/ml. The saline treated ob/ob mice averaged 1.19ng/ml, with a range of 1.01 - 1.34 ng/ml. This background may be due to reactivity with the truncated leptin protein which is the predicted product of the ob mutation. At week 7, the same groups of mice and five C57 control mice were tested.
  • Serum leptin levels in the rAAV leptin treated mice decreased to 2.46 ng/ml at week 9.
  • Untreated ob/ob mice had circulating leptin levels of 65 ng/ml and the wild-type C57 mice had levels of 4.19 ng/ml at this timepoint.
  • the leptin concentration was 2.97 ng/ml in treated mice versus 1.03 ng/ml of reactive protein in the untreated ob/ob mice. This is again in the range of normal C57 black mice (2.31 ng/ml).
  • P values for treated versus untreated are .09, .0005, 007 and .0079 for weeks 5, 7, 9 and 11, respectively.
  • ectopic expression of physiologic levels of leptin can prevent onset of obesity.
  • the RIA employed in this study also detects some activity in untreated ob/ob mice serum. This might be due to the presence of endogenous inactive leptin secreted in this strain of mice (the ob defect is due to premature termination codon in the leptin coding sequence).
  • the ob/ob phenotype is characterized by insulin-resistant diabetes; ob/ob mice are hyperglycemic, despite elevated levels of circulating insulin, to determine the effects of leptin gene therapy on diabetes, fasting blood glucose and insulin were measured. Mice were fasted for eighteen hours and bled for determination of fasting glucose (Figure 5 A) and insulin (Figure 5B), six weeks post-injection. The values presented here are the mean _+ SEM of five mice.
  • Figure 5C Glucose tolerance as determined in saline ( ⁇ ), r-AAV-leptin treated (O), or C57(*) mice by measuring blood glucose levels at indicated times after intraperitoneal injection of glucose.
  • AAV-leptin treated mice to clear glucose from circulation.
  • a bolus of lmg/gm glucose was injected i.p. into fasted mice and blood glucose was monitored over time.
  • the level of circulating glucose peaked at 30 minutes and returned to normal within 120 min ( Figure 5C).
  • the level of glucose was at least twofold greater than the leptin treated mice at all timepoints. The glucose levels in these mice did not normalize within the three hour time course of the study.
  • hyperinsulinemia and insulin resistance could be corrected in leptin treated mice.
  • FIG 5 at week 6 there was a complete reversal of hyperinsulinemia and hyperglycemia in treated animals.
  • the levels of circulating insulin in treated animals were similar to levels reported for C57 mice (.54 ng/ml versus .40 ng/ml).
  • rAAV-leptin treated mice had a normal response to a glucose challenge.
  • control ob/ob mice failed to correct the exaggerated hyperglycemia state after a post-fast injection of glucose.
  • leptin-treated and age-matched C57BL mice corrected their hyperglycemia.
  • pKm201CMV is an AAV cloning vector in which an expression cassette, consisting of a CMV immediate early enhancer, promoter and intron, and a bovine growth hormone (bGH) polyadenylation site, is flanked by inverted terminal repeat (ITR) sequences from AAV-2.
  • pKm201CMV was derived from pKm201, a modified AAV vector plasmid in which the ampicillin resistance gene of pEMBL-AAV-ITR (see Srivastava, (1989) Proc. Natl. Acad. Sci. USA 86, 8078-8082) has been replaced with the gene for kanamycin resistance.
  • the expression cassette from pCMVlink a derivative of pCMV ⁇ c (see Chapman, (1991) Nucleic Acids Res.19, 193-198) in which the BGH poly A site has been substituted for the SV40 terminator, was inserted between the ITRs of pKm201 to generate pKm201CMV.
  • the Avr II - Bglll fragment which encodes the full length monkey Epo sequence, from the cline pMKE83 (ATCC Accession Number 67545) we cloned into the Xba I - BamH I sites of pKm201 CMV.
  • the AAV vector contains a post-transcriptional regulatory element (PRE) from hepatitis B virus.
  • the PRE which increases efficiency of mRNA transport (see Huang, Mol. Cell. Bio. 15, 3864-3869), was included to increase the size of the vector genome for more efficient packaging.
  • a 579 bp fragment, from the post-transcriptional regulatory element (PRE) region of Hepatitis-B (HBV) was amplified using the primer set:
  • the resulting fragment was digested with As I and Mlu I and inserted into an Mlu I site between the leptin coding region and the BGH poly A site. Inclusion of the coding region for mouse leptin into this construct results in a 3.4 kb packageable vector genome.
  • This vector plasmid was packaged using standard methods, and a purified stock of 1.25xl0 12 particles/ml was obtained.
  • AAV helper plasmid pKSrep/cap (encoding rep and cap protein) was constructed by cloning the AAV-2 genome, without the ITRs (AAV-2 nucleotides 192 through 4493) into pBluescript II KS+ (Stratagene, La Jolla, CA).
  • Recombinant AAV-Epo particles were produced and analyzed following the protocols in Examples 2 and 3, except that HT1080 cells (human fibrosarcoma cells) maintained in DME+ 10% fetal calf serum (FCS), plated (2xl0 5 cells) on a 6 well dish the day before infection were used.
  • the cells were infected with rAAV-Epo at different MOI and 48 hours later supernatant was monitored for Epo using the R&D Quantikine ELISA kit (R&D Systems, Minneapolis, MN). The ELISA results are shown in Fig. 2. Titers of rAAV-Epo are indicated on the X-axis.
  • the lane marked leptin represents background levels of Epo secreted from cells infected with 5 x 10 9 particles of rAAV-m-leptin.
  • the results show that infection of HT1080 cells with 5 x 10 9 particles produced 10,800 mIU/10 6 cells/day, which is equivalent to 86.4 ng of Epo per day (1 mlU is equivalent to ⁇ 8 pg of Epo), while infection with 2 x 10 8 and 1 x 10 9 particles led to production of 480 and 2200 mIU/10 6 cell/day, respectively.
  • EXAMPLE 6 In Vivo Administration of rAAV-Epo In Mice
  • mice Seven week old female C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). Recombinant AAV-Epo was administered by injection of 50 ⁇ l of normal or normal saline containing 2 x 10 u rAAV particles into the TA muscle of mice anesthetized with a mixture of ketamine and xylazine as described in Example 4.
  • Panel A shows serum Epo concentration (+/-S.E.M.) following rAAV administration. At all timepoints, the saline injected mice had undetectable levels of serum Epo and are not included in the Figure.
  • Panel B shows the average hematocrit of four mice injected with either rAAV-Epo or saline. Although error bars are included, they are obscured by the plot symbols. The 0 week timepoints represent the average baseline hematocrit and serum Epo concentrations for untreated C57BL/6 mice.
  • a bioluminescent ELISA assay was developed to detect antibodies against Epo.
  • Microtiter plates (Dynatech Microlite, Chantilly, VA) were coated with 50 ⁇ l of EPOGEN ® (h-erythropoietin, Amgen, Thousand Oaks, CA) in a 1 ⁇ g/ml solution in PBS and incubated overnight at 4°C or for one hour at 37°C.
  • EPOGEN ® h-erythropoietin, Amgen, Thousand Oaks, CA
  • the coated wells were blocked with lx Aqualite Streptavidin Assay Buffer (Sealite Sciences, Boggard, GA) containing 5 % goat serum for one hour at 37°C.
  • the plates were then washed three times with lx Aqualite Washing Buffer containing 1 % goat serum and 3 % Tween-20. Diluted serum samples (50 ⁇ l) were transferred onto coated plates and incubated at 37°C for one hour, then washed six times with washing buffer. Primary antibody (goat anti-mouse gig from Sigma) diluted to 1: 1000 was added and incubated for one hour followed by washing six times. Streptavidin Aqualite antibody (1:500) was added to each well and incubated at 37°C for one hour followed by washing six times. The luminescence was triggered by injections of 50 ⁇ l aliquots of lx Trigger buffer and the plates were read with a Dynatech ML3000 Luminometer (Chantilly, VA).
  • mice For saline treated mice, sera was pooled prior to ELISA, sera from rAAV treated mice were measured individually. Positive control (+ control) represents sera from cynomolgous monkey-Epo plasmid DNA injected BALB/c mice which had previously been shown to have anti-cm-Epo antibodies. Titer is defined as the dilution of serum required to reduce the signal to levels obtained in wells containing dilution buffer alone.
  • Figs. 9A-9B The results are shown in Figs. 9A-9B.
  • Fig. 9 A shows the plasma
  • Fig. 9B shows the hematocrits of two baboons at time pre-injection (negative numbers) and post-injection.
  • 34 or 40 ml of blood was removed from Baboon 2 at weeks 11 and 13.
  • pre-injection values for serum Epo ranged from 1.5 - 3.3 mlU/ml, with minor week to week variation.
  • Epo levels had increased to 4.5 mlU/ml for Baboon I and to over mlU/ml for Baboon 2.
  • week 4 values had increased to 11.8 mlU/ml for Baboon 1 and to 11.3 mlU/ml for Baboon 2.
  • Values peaked at weeks 8-10, at which time Baboon I had circulating levels of 35.9 mlU/ml and Baboon 2 had circulating levels of 41.6 mlU/ml.
  • the hematocrit of Baboon 2 ranged from 38.7 to 42.3 % .
  • the hematocrits of both baboons did not increase significantly during the first week. This probably reflects the lag between exposure to Epo and differentiation of precursor cells into erythrocytes.
  • the hematocrits of both baboons had increased above the range seen prior to injection. Hematocrits continued to increase until week 10, at which time both baboons showed at least 25 point increases over their pre-treatment levels.
  • the hematocrit of Baboon 2 exceeded 70%; and thus, this animal was phlebotomized o reduce the risk of thrombosis.
  • a solution of rAAV-Epo virions at a concentration of 1 x 10 12 in 0.9% NaCI is placed in the reservoir of a Ultravent nebulizer (Mallinckrodt) .
  • the nebulizer is driven at 40 psi with compressed air.
  • the size distribution of aerosol droplets is determined by laser particle-size analysis and the relative proportion of the virion preparation is evaluated by collecting the aerosolized droplets in phosphate buffered saline, pH 7.4, as described in Hubbard, Proc. Natl. Acad Sci 86, (1989) 680-684. Seven week old female C57BL/6 mice are obtained from Charles
  • Blood (200 ⁇ l) is collected by using retro-orbital bleeds following isoflurane edition. Whole blood is used for hematocrit estimation and separated serum is used for detecting the Epo by ELISA.
  • a bioluminescent ELISA assay can be employed to detect antibodies against Epo.
  • Microtiter plates (Dynatech Microlite, Chantilly, VA) are coated with 50 ⁇ l of EPOGEN ® (h-erythropoietin, Amgen, Thousand Oaks, CA) in a 1 ⁇ g/ml solution in PBS and incubated overnight at 4°C or for one hour at 37°C.
  • EPOGEN ® h-erythropoietin, Amgen, Thousand Oaks, CA
  • the coated wells are blocked with lx Aqualite Streptavidin Assay Buffer (Sealite Sciences, Boggard, GA) containing 5% goat serum for one hour at 37°C.
  • the plates are then washed three times with 1 x Aqualite Washing Buffer containing 1 % goat serum and 3 % Tween-20. Diluted serum samples (50 ⁇ l) ate transferred onto coated plates and incubated at 37°C for one hour, then washed six times with washing buffer.
  • Primary antibody (goat anti-mouse IgG from Sigma) diluted to 1: 1000 is added and incubated for one hour followed by washing six times.
  • Streptavidin Aqualite antibody (1:500) is added to each well and incubated at 37°C for one hour followed by washing six times.
  • the luminescence is triggered by injections of 50 ⁇ l aliquots of 1 x Trigger buffer and the lates are read with a Dynatech ML3000 Luminometer (Chantilly, VA). Titer is defined as the dilution of serum required to reduce the signal to levels obtained in wells containing dilution buffer alone.
  • equivalent human dosages by weight should be used, for example from 1 x 10 7 to 1 x 10 16 particles in 50 ⁇ l volumes.
  • Intra-nasal formulations of rAAV virions carrying other heterologous sequences can be made in accordance with the methods disclosed herein, for example rAAV-leptin, and administered and tested as described above and employing art recognized methods.
  • Such formulations may be administered in humans in a fashion analogous to administration in mice, for example, via intra-nasal instillation using an Ultravent (Mallinckrodt) aerosol nebulizer in accordance with the manufacturer's instructions.
  • Ultravent Meallinckrodt aerosol nebulizer
  • C57BL/6 mice Six week old female C57BL/6 mice were purchased from Charles River Labs (Wilmington, MA). C57BL/6 class I deficient and C57BL/6 class II deficient mice were purchased from Taconic Labs (Germantown, NY). CD40 ligand deficient mice and B129 mice were purchased from Jackson Labs (Bar Harbor, ME).
  • Vector pAAV-lacZ was constructed by cloning the LacZ expression cassette from pCMV- ⁇ (Clontech, Palo Alto, CA) containing the CMV promoter, intron, LacZ and SV40 polyadenylation signal into pEMBL-AAV-ITR.
  • Plasmid pkm201 is a derivative of pEMBL-AAV-ITR in which the ampicillin resistance gene was replaced with the gene for kanamycin resistance. See Example 1.
  • Plasmid pAAV-Luc was constructed by cloning an expression cassette containing the CMV promoter/intron, luciferase and the bovine growth hormone polyadenylation signal into pKm201.
  • Plasmid pKSrep/cap was constructed by cloning the AAV-2 genome, without the ITRs (AAV -2 nucleotides 192 through 4493) into pBluescript II KS+ (Strategene, La Jolla, CA). See Example 1.
  • Recombinant AAV particles were produced as disclosed in Example 2. Residual adenovirus contamination was inactivated by heating at 56°C for 45 min. To estimate total number of rAAV particles, the stock was treated with DNAse I and encapsidated DNA was extracted with phenol-chloroform and precipitated with ethanol. DNA dot blot analysis against a known standard was used to determine titer. To assay for adenovirus contamination, 293 cells were infected with lO ⁇ l of purified rAAV stock and followed for any signs of cytopathic effect. All stocks were negative, indicating that adenovirus contamination was less than 100 pfu/ml.
  • the rAAV particles were diluted in 0.9% saline and a final volume of 50 ⁇ l was injected into the tibialis anterior (TA) muscle.
  • groups of five class I knockout, class II knockout or C57BL/6 mice were injected with 1 x 10 10 particles rAAV -LacZ in the right TA.
  • the mice were bled for serum and injected with either 1 x 10 10 particles rAAV- LacZ (three animals) or 1 x 10 10 particles of rAAV-Luc (two animals) in the left TA.
  • the mice were bled again, sacrificed and muscles were collected and immediately frozen in liquid nitrogen for either LacZ staining or luciferase assay.
  • mice were injected with lOO ⁇ g rat anti-mouse CD4 (clone GK1.5, Pharmingen, San Diego, CA) by intraperitoneal injection at days -3, 0 and +3 relative to the first injection (at day 0) of rAAV.
  • mice received lOO ⁇ g of antibody (clone MRl, Pharmingen, San Diego, CA) by intraperitoneal injection at days -3, 0 and +3 and +6 relative to the first injection of rAAV.
  • mice treated with cyclosporin A received intraperitoneal injections of 10 mg/kg drug every five days from one week before the first injection of rAAV until the termination of the experiment.
  • the frozen muscles were ground in a pre- chilled mortar and pestle, transferred to a 1.5 ml micro fuge tube and resuspended in 500 ⁇ l lx reporter lysis buffer (Promega, Madison, WI). The tubes were vortexed for fifteen minutes at room temperature and then freeze/thawed three times. Ly sates were cleared by centrifuging at maximum speed in a micro fuge for ten minutes and then stored at -80°C until assayed.
  • Luciferase assays were performed using the manufacturer's protocol (Promega, Madison, WI) and read on a Dynatech ML3000 (Chantilly, VA) plate luminometer. Protein concentration of each of the lysates were assayed by BCA protein assay (Pierce, Rockford, IL) and luciferase activities were expressed as picograms of luciferase per mg protein. Cryosections (8 ⁇ m) were fixed for five minutes at room temperatures in 10 mM phosphate buffered saline (PBS) containing 1 % paraformaldehyde.
  • PBS phosphate buffered saline
  • the fixed sections were stained with X-gal solution (PBS containing 1 mg/ml 5-bromo-4-chloro-3-indoyl- ⁇ -galactopyranoside, 1 mM MgCl 2 , 5 mM K 3 Fe(CN) 6 , and 5 mM K 4 Fe(CN) 6 ) for sixteen hours at 37°C. Sections were counterstained with Nuclear Fast Red.
  • X-gal solution PBS containing 1 mg/ml 5-bromo-4-chloro-3-indoyl- ⁇ -galactopyranoside, 1 mM MgCl 2 , 5 mM K 3 Fe(CN) 6 , and 5 mM K 4 Fe(CN) 6
  • microtiter plates were coated overnight at 4°C with 10 9 rAAV-LacZ particles/well in PBS. The following day, the plates were washed and then blocked for thirty minutes at 37°C with PBS containing 1 % goat serum and 0.3 % Tween 20. Serial three-fold dilutions of sample and control sera were loaded onto the plate starting at 1 :75 (control sera was from mice which had received multiple injections of AAV). The microtiter plates were then incubated for one hour at 37°C.
  • Plates were washed and incubated at 37°C for thirty minutes with goat anti-mouse Ig-HRP (immunoglobulin labeled with horseradish peroxidase) at 1:2000 (Dako, Carpenteria, CA). O-phenylenediamine substrate was used to develop the plates. Plates were read at 492 nm with a cut-off of 0.2 OD.
  • Ig-HRP immunoglobulin labeled with horseradish peroxidase
  • AAV neutralizing antibody titer was defined as the dilution of serum required to see 50% of the luciferase activity in 293 cells infected with rAAV-Luc pre-incubated with negative control serum.
  • CD40L CD40 ligand
  • CD40L is expressed on activated CD4+ T cells and is critical for their ability to provide help to B cells.
  • CD40 ligand deficient mice are known to be deficient in humoral immune responses.
  • An experiment identical to the experiment described above was performed in CD40 ligand deficient mice, except that in all mice the second injection was rAAV-Luc and B129 mice were used as the control. The results are shown in Table 2 below. NA stands for not applicable, and ND stands for not determined.
  • readministration of rAAV was not possible in the wild type control mice (B129) due to the high anti-AAV titers, but possible in the CD40 ligand deficient mice.
  • the ability to obtain recombinant protein (i.e., luciferase) expression upon vector readministration correlated inversely with anti- AAV antibody titer and AAV neutralizing antibody titer.
  • mice As shown in Table 3, the groups that received the lower doses (10 4 , 10 5 ), were as efficiently re- injected as the native controls. These same groups of mice also did not demonstrate neutralizing antibody responses to AAV indicating that the amount of antigen in these doses may have been too low to elicit an immune response. In fact, the right TA muscles of these mice were all negative for lacZ expression (data not shown).
  • the group that received 10 8 particles of rAAV-lacZ mounted a weak antibody response to AV. This lower antibody response resulted in an intermediate level of luciferase expression from the second injection. In this group, one of the two animals with measurable neutralizing antibody titers showed the lowest luciferase expression. The group receiving 10 10 particles demonstrated a robust antibody response to AV and second administration was not successful.
  • mice treated with anti-CD4 antibody at the time of first injection were able to be re-injected with rAAV-Luc.
  • Formulation and injection protocols were as previously described. All injections were 1 x 10 10 particles rAAV unless otherwise noted. Luciferase activity was measured and expressed in picograms luciferase per mg protein. The results are shown in Table 4 below. ND stands for not determined.
  • CD40L knock-out mice were performed in CD40L knock-out mice.
  • CD40L is expressed on activated CD4 + T cells and is critical for their ability to provide help to B cells.
  • the CD40L knock out mice should mimic the responses seen in the Class II knock out mice.
  • the rAAV mediated transgene expression levels in this strain of control mice (B129) was much lower than in the C57B1/6 control mice, demonstrating that mouse haplotype can also influence the expression levels mediated by rAAV vectors.
  • mice Seven week old female C57BL/6 mice are obtained from Charles River Laboratories (Cambridge, MA). After anesthetization with a mixture of ketamine and xylazine and baseline blood samples are obtained, a mid-abdomen incision is made and the ureter and uretropelvic junction are freed of connective tissues and vascular structures to expose the left renal artery, which is then clamped. An aliquot of 50 ⁇ l of a 5 % Dextrose solution containing 10 8 to 10 12 particles of rAAV-Epo or of rAAV-leptin, produced as described in Examples 1-4 above, is directly infused into the left renal artery using a 30 gauge needle within 1 minute.
  • the renal blood flow is then re-established, 5 minutes after injection by removal of the clamp. The incision is closed and the animals allowed to recover. See Lai, Gene Therapy 4 (1997) 426-31 and Yamada, J Clin Invest 96 (1995)1230- 37. Three days after injection, blood samples are collected for analysis. Whole blood is used for hematocrit estimation and separated serum is used for detecting the Epo by ELISA.
  • a bio luminescent ELISA assay can be employed to detect antibodies against Epo.
  • Microtiter plates (Dynatech Microlite, Chantilly, VA) are coated with 50 ⁇ l of Epogen (Epotin, Amgen, Thousand Oaks, CA) in a 1 ⁇ g/ml solution in PBS and incubated overnight at 4°C or for 1 hour at 37 °C.
  • Epogen Epogen
  • the coated wells are blocked with lx Aqualite Streptavidin Assay Buffer (Sealite Sciences, Boggard, GA) containing 5 % goat serum for 1 hour at 37 °C.
  • the plates are then washed 3 time with lx Aqualite Washing Buffer containing 1 % goat serum and 3 % Tween-20. Diluted serum samples (50 ⁇ l) are transferred onto coated plates and incubated at 37 °C for one hour, then washed 6 times with washing buffer. Primary antibody (goat anti ouse IgG from Sigma) diluted to 1: 1000 is added and incubated for 1 hours followed by washing 6 times. Streptavidin Aqualite antibody (1:500) is added to each well and incubated at 37 °C for one hour followed by washing 6 times.
  • the luminescence is triggered by injections of 50 ⁇ l aliquots of lx Trigger buffer and the plates are read with a Dynatech ML3000 Luminometer (Chantilly, VA). Titer is defined as the dilution of serum required to reduce the signal to levels obtained in wells containing dilution buffer alone.
  • mice infused with rAAV-leptin For mice infused with rAAV-leptin, the animals are fasted for 18 hours and blood collected from the tail vein to determine fasting glucose levels. The mice then receive 1 mg/g body weight of a sterile glucose solution by i.p. injection. The mice are anesthetized with isoflurane and blood samples are collected via retroorbital bleeds at 15, 30, 60, 120 and 180 min following the injection. Circulating glucose is measured using the Lifescan One Touch monitor (Life Scan, Milpitas, CA). Insulin levels are measured with the Linco Rat Insulin RIA kit (Linco Research Immunoassay, St. Charles, MO). Leptin levels are measured with the Lincomouse Leptin RIA kit.
  • Formulations of rAAV virions for direct injection into the renal artery carrying other heterologous sequences can be made in accordance with the methods disclosed here and administered and tested as described above and employing art recognized methods.
  • Formulations of rAAV virions for human administration via direct injection into the renal artery are made and administered in a manner similar to that described above.
  • Human doses equivalent (by weight) to the doses employed in mice can be used.
  • Administration can be effected by modification of the medical technique of angiography. As is well known in the art, in angiography a catheter is inserted into the femoral artery of the patient and dye injected in order to visualize the kidney.
  • a catheter is inserted into the femoral artery and a pharmaceutical composition comprising a sterile solution containing an effective amount of a rAAV virion in admixture with a pharmaceutically acceptable carrier is injected.
  • a pharmaceutical composition comprising a sterile solution containing an effective amount of a rAAV virion in admixture with a pharmaceutically acceptable carrier.

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Abstract

L'invention concerne un procédé permettant d'appliquer à un patient une thérapie génique dans laquelle le médiateur est un vecteur viral adéno-associé. Le procédé consiste à administrer à un patient une particule de virus adéno-associé déficiente du point de vue de la réplication qui infecte une cellule du patient, la particule contenant un gène qui code une protéine nécessaire au patient, le gène étant lié de manière efficace pour l'expression dans la cellule; le procédé consiste également à administrer à peu près au moment de l'étape précédente, un immunosuppresseur qui supprime la réponse immunitaire humorale du patient. Cette invention concerne également des compositions pharmaceutique contenant le virus adéno-associé décrit précédemment et l'immuno-suppresseur humoral dans un support pharmaceutiquement acceptable. Des exemples de protéines exprimées par les vecteurs décrits précédemment comprennent l'erythropoïétine, la thrombopoïétine, le facteur de croissance humaine, la leptine, le facteur VIII, le facteur IX, le facteur Xa et autres.
PCT/US1998/015794 1997-07-31 1998-07-29 Procede permettant de readministrer un vecteur viral adeno-associe via l'immunodepression de l'hote WO1999006562A1 (fr)

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JP2000505303A JP2001512142A (ja) 1997-07-31 1998-07-29 宿主の免疫抑制を介するａａｖベクターの再投与を可能にする方法
AU86721/98A AU8672198A (en) 1997-07-31 1998-07-29 Method enabling readministration of aav vector via immunosuppression of host
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US10538571B2 (en) 2017-11-27 2020-01-21 Coda Biotherapeutics, Inc. Compositions and methods for neurological diseases
US12215338B2 (en) 2018-05-08 2025-02-04 Neuracle Science Co., Ltd. Adeno-associated virus (AAV) delivery of anti-FAM19A5 antibodies
WO2020160508A1 (fr) 2019-01-31 2020-08-06 Oregon Health & Science University Méthodes d'utilisation d'une évolution dirigée, dépendant d'une transcription, de capsides aav
WO2021035179A1 (fr) 2019-08-21 2021-02-25 Coda Biotherapeutics, Inc. Compositions et méthodes de traitement de maladies neurologiques
WO2023212294A1 (fr) 2022-04-29 2023-11-02 Broadwing Bio Llc Anticorps spécifiques de la protéine 7 liée à l'angiopoïétine et leurs utilisations
WO2023212298A1 (fr) 2022-04-29 2023-11-02 Broadwing Bio Llc Anticorps bispécifiques et méthodes de traitement d'une maladie oculaire
WO2023212293A1 (fr) 2022-04-29 2023-11-02 Broadwing Bio Llc Anticorps spécifiques 4 associés au facteur h du complément et leurs utilisations
WO2024227154A1 (fr) 2023-04-28 2024-10-31 Broadwing Bio Llc Anticorps spécifiques de composant 3 du complément (c3) et leurs utilisations

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EP1002078A1 (fr) 2000-05-24
AU8672198A (en) 1999-02-22
JP2001512142A (ja) 2001-08-21

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