+

WO1997039776A9 - Therapie genique reposant sur l'administration conjointe et repetee d'adenovirus et d'agents immunosuppresseurs - Google Patents

Therapie genique reposant sur l'administration conjointe et repetee d'adenovirus et d'agents immunosuppresseurs

Info

Publication number
WO1997039776A9
WO1997039776A9 PCT/US1997/006199 US9706199W WO9739776A9 WO 1997039776 A9 WO1997039776 A9 WO 1997039776A9 US 9706199 W US9706199 W US 9706199W WO 9739776 A9 WO9739776 A9 WO 9739776A9
Authority
WO
WIPO (PCT)
Prior art keywords
vector
administration
mice
cells
adenoviral
Prior art date
Application number
PCT/US1997/006199
Other languages
English (en)
Other versions
WO1997039776A1 (fr
Filing date
Publication date
Application filed filed Critical
Priority to AU26688/97A priority Critical patent/AU2668897A/en
Publication of WO1997039776A1 publication Critical patent/WO1997039776A1/fr
Publication of WO1997039776A9 publication Critical patent/WO1997039776A9/fr

Links

Definitions

  • This invention relates to gene therapy comprising the use of adenoviruses as the gene delivery vehicles . More particularly, this invention relates to gene therapy involving the concurrent and repeated administration of adenoviruses and immunosuppressive agents, whereby the efficiency of the gene therapy treatment is enhanced through suppression of an immune response against the adenoviruses.
  • Adenoviruses can be very efficient in gene transfer into cells in vivo, and, thus may be employed as delivery vehicles for introducing desired genes into eukaryotic cells, whereby the adenovirus delivers such genes to eukaryotic cells by binding cellular receptors.
  • adenovirus gene transfer There are, however, several limitations to adenovirus gene transfer which are due in part to host responses directed at either the adenovirus vector particle, breakdown products of the vector particle, or the transduced cells. These host responses include non-specific responses and specific immune responses. The non-specific responses include inflammatory and non-inflammatory changes. An example of the latter is a change in host cell gene expression.
  • Specific immune responses include various cellular responses and humoral antibody responses. Cellular responses include those mediated by T-helper lymphocytes, T- suppressor lymphocytes, cytotoxic T lymphocytes (CTL) , and natural killer cells.
  • T-cell response contributes to, but is not responsible solely for, the limited duration of expression in adults from adenovirus vectors.
  • the authors further show that cyclosporin A is not effective in blocking the humoral response to the vector.
  • Figure 3 is a schematic of the construction of plasmid pAvS6;
  • Figure 4 is a map of plasmid pAvS6;
  • Figure 5 is a map of plasmid pBQ4.7 ;
  • Figures 10A, 10B, and IOC depict the histologic appearance of the lung in response to AvlCf2 administration three days after vector administration;
  • Figure 12 is a graph of anti-adenoviral antibody titers of lung lavage samples from rats infected with AvlCf2 and which were treated or not treated with dexamethasone;
  • Figure 13 is a graph of CTL responses in rats 42 days after infection with AvlCf2 ;
  • Figure 15 is a map of plasmid pAvlH9FR;
  • Figure 16 is a schematic of the adenoviral vector AvlH9F2 ;
  • a method of effecting a gene therapy treatment in a host comprises administering to a host (i) an adenoviral vector including at least one DNA sequence and (ii) an immunosuppressive agent.
  • the course of administration of the adenoviral vector and immunosuppressive agent then is discontinued.
  • Administration of the immunosuppressive agent and the adenoviral vector then is repeated at least once.
  • the adenoviral vector is administered in an amount effective to produce a therapeutic effect in the host.
  • the immunosuppressive agent is administered in an amount effective to prevent or suppress a humoral and/or cellular immune response to the vector and/or cells containing the vector.
  • Agents which affect humoral antibody responses are generally directed at antibody producing B lymphocytes (B- cells) or at the T-cells which are responsible for inducing B-cell antibody production to high levels.
  • the ITR, encapsidation signal, Rous Sarcoma Virus promoter, the adenoviral tripartite leader (TPL) sequence and linking sequences were assembled as a block using PCR amplification ( Figure 2) .
  • the ITR and encapsidation signal (sequences 1-392 of Ad-dl327 [identical to sequences from Ad5, Genbank accession #M73260] incorporated herein by reference) were amplified (amplification 1) together from Ad-dl327 using primers containing NotI or Ascl restriction sites.
  • Sensitizer cells were prepared by infecting cotton rat lung fibroblasts with Ad-dl327 at a multiplicity of infection of 100. The cells were incubated for 3 days, and checked for hexon expression by FACS . The cells then were washed with PBS/EDTA, contacted with trypsin, washed, spun, and resuspended in 1 ml RPMI medium. The cells then were irradiated with 13'7 Cs at 5,000 rads in order to inactivate the DNA.
  • Spleens then were isolated from uninfected (control) rats and adenovirus-infected rats 42 days after infection.
  • the spleens were kept in sterile HBSS and ice. 10 ml of HBSS then was injected into each spleen with a 25/27 gauge needle. The spleen was mashed, and filtered with a cell strainer into a 50 ml tube. The volume then was brought to 40 ml in RPMI plus 10% FCS. The tube was spun at 1,500 rpm for 10 minutes. Red blood cells then were lysed by adding 2.5 ml of ACK lysis buffer, and the liquid was swirled for less than 1 minute. The volume was brought up to 50 ml with RPMI-10.
  • AvlH9F2 is constructed from a derivative of the adenoviral shuttle plasmid vector pAvlH9FR ( Figure 15) , which includes human Factor IX DNA, and is described in PCT application No. W094/29471, published December 22, 1994.
  • Neutralizing antibody titers in 20 mice are given in Table I below. As indicated in Table I, DSG is deoxyspergualin, Cy is cyclophosphamide, and Dex is dexamethasone .
  • AvlALAPH ⁇ l is an adenoviral vector which co ⁇ Ui WS tkiQ &* domain deleted human Factor VIII cDNA expressed from the mouse albumin promoter, and is described in published PCT Application No. W094/29471.
  • Deoxyspergualin is an immunosuppressant currently being tested clinically in organ transplantation. It has a potent, long term effect on antigen specific B cells and has been shown to prevent effectively the production of specific antibody when co-administered with protein antigens.
  • Alegre, et al . , Transplantation, Vol. 57, pgs. 1786-1794 (1994); Tepper, Ann. N. Y. Acad. Sci .. pgs. 123-132 (1993); Tufveson, et al . , Transplant . Proc .. Vol. 26, pgs. 3029-3039 (1994) .
  • the mode of action of DSG is not fully understood at the molecular level .
  • Plasma levels of human Factor VIII were determined by ELISA, as described in Connelly, et al . , Human Gene Therapy, Vol. 6, pgs. 185-193 (1995). The limit of sensitivity with mouse plasma samples containing B domain deleted human Factor VIII was 3 to 6 ng/ml. Mouse plasma samples were diluted 1:5 prior to the assay, therefore, the actual limit of detection was 15 to 30 ng/ml.
  • Plasma levels of human Factor IX were determined by ELISA. Asserachrom IX:Ag ELISA kits were purchased from American Bioproducts Company (Parsippany, NJ) and assays were performed according to the manufacturer's instructions. The limit of sensitivity was 1.6 ng/ml.
  • Mouse plasma or serum samples were heat inactivated at 55 °C for 30 minutes and then diluted in Improved Minimal Essential Medium (Biofluids, Rockville, MD) plus 2% FBS (IMEM/2%FBS) in two-fold steps beginning at 1:2.
  • 55 ⁇ l of each sample were mixed with 10 ⁇ l of AvllacZ4 (containing 4 x 10 5 pfu) , incubated for 1 hour at 37 °C and applied to nearly confluent 293 cells in 96 well plates (4 x 10 4 cells per well) . After 60 minutes in the tissue culture incubator, the virus was aspirated from each well and replaced with 150 ⁇ l of IMEM/10%FBS.
  • adenovirus vector AvllacZ4 to C57BL/6 mice via tail vein were administered.
  • the vector inoculum ranged from 1 x 10 3 pfu to 1 x 10 8 pfu in single log increments.
  • serum levels of anti-adenovirus neutralizing antibodies were determined (Fig. 18) for mice which received 1x10 s pfu or greater of vector. A minus sign indicates that none of the mice in the cohort had detectable antibody.
  • C57BL/6 mice were immunosuppressed with either deoxyspergualin (DSG) , cyclophosphamide (Cy) , or dexamethasone (Dex) at the time of administration of 1 x 10 ⁇ pfu of AvllacZ4. As shown above, this dose completely prevented an effective second delivery. Mice were injected daily with 33 mg/kg of DSG, beginning one day before vector delivery and continuing for seven more days. Dexamethasone was delivered over the same time course, at a dose of 5 mg/kg. Cyclophosphamide was administered once, the day before vector delivery, at a dose of either 100 mg/kg or 300 mg/kg. Control mice received AvllacZ4 without immunosuppression, or were immunosuppressed without initial vector delivery.
  • DSG deoxyspergualin
  • Cy cyclophosphamide
  • Dex dexamethasone
  • each mouse received 1 x 10 8 pfu of AvlH9F2.
  • human Factor IX plasma levels were determined by ELISA (Fig. 21) .
  • Control mice which were not pre-immunized with AvlLacZ4, expressed an average of 9 ⁇ g/ml of human Factor IX.
  • Other control mice which received AvllacZ4 but were not immunosuppressed, expressed no human Factor IX after AvlH9F2 administration.
  • the one mouse which was immunosuppressed with 33 mg/kg DSG expressed 12 ⁇ g/ml of human Factor IX.
  • DSG does not produce a general suppression of the immune system, but rather results in a selective lack of response to specific antigens presented at the time of drug treatment .
  • immunosuppression with DSG over a 7 day period following vector delivery efficiently inhibited the humoral response to the vector and permitted an effective second administration.
  • the initial experiment with DSG employed a high dose of 33 mg/kg, which is close to the maximum tolerated dose in mice and several fold higher than the doses used in human trials .
  • lower doses of DSG were also effective in permitting repeat delivery of vector.
  • a greater degree of individual variability in levels of Factor IX expre ⁇ ion was seen with reduced doses, although even at the lowest dose tested (5 mg/kg) significant Factor IX expression was obtained in 3 of 6 animals .
  • Cyclophosphamide administered at a dose of 300 mg/kg the day before vector injection, was also effective in blocking the humoral response and allowed a completely effective second injection with a Factor IX adenovirus vector. Furthermore, a third injection with a Factor VIII encoding vector was also completely efficacious when the previous two vector administrations were each preceded by a single dose of cyclophosphamide. Cyclophosphamide is used clinically as an anti-cancer agent in the treatment of Hodgkins disease and other leukemias . It is also employed as an immunosuppres ⁇ ive agent in the treatment of hemophilia patients who develop inhibitors to Factor VIII protein replacement therapy. (Aledort, Am. J . Hemat .. Vol.
  • mice While the dose used to successfully obtain readministration in mice is substantially higher than is generally used in humans, it remains to be established whether lower, clinically acceptable doses, might be effective in humans.
  • One possibility suggested by experience in the organ transplantation setting is that combinations of immunosuppressants would yield more potent suppression of the immune system with less toxicity.
  • cyclophosphamide may be effective at lower dose ⁇ when u ⁇ ed in combination with dexamethasone.
  • AvlH9F2 used in this study, 1 x 10 8 pfu, yielded plasma levels of human Factor IX of 5-10 ⁇ g/ml, which is 20 to 50 times above a level that would be therapeutic in a hemophiliac.
  • Vectors such as AvlH9F2, which express high levels of transgene product and which can be administered at relatively low doses, should reduce the extent of immune stimulation and the degree of immunosuppression required.
  • This example describes transient suppression of the humoral immune system with a combination of DSG and cyclophosphamide.
  • the data presented in this example demonstrate that these two drugs can be used in combination at clinically relevant doses to inhibit a humoral response directed against an adenoviral vector and thus allow multiple repeat administrations of vector.
  • the adenoviral vector employed in this example was Av3nLacZ.
  • Av3nLacZ is described in Example 2 of U.S. Patent Application Serial No. 08/458,403, entitled "Improved Adenoviral Vectors and Producer Cells," filed June 2, 1995, the contents of which are incorporated herein by reference.
  • mice treated in this example and a general outline of the treatment regimen for each cohort are given in Table II below.
  • mice which received a first injection of Av3nLacZ without immunosuppression received a second injection of 1 x 10 8 pfu of Av3nLacZ without immunosuppression (cohort 4) .
  • All of the mice which received AvlH9F2 were bled one week after vector admini ⁇ tration and the plasma levels of human Factor IX were determined by ELISA. The results are shown in Figure 22.
  • mice which received Av3nLacZ without immunosuppression all had undetectable levels of human Factor IX one week after AvlH9F2 delivery.
  • the results corresponding to these mice are designated as "2 nd administration" in Figure 22.
  • immunosuppre ⁇ sion with a combination of DSG and cyclophosphamide enabled a completely efficacious second administration of vector.
  • mice which received two injections of Av3nLacZ were bled five weeks after the second vector administration and plasma levels of anti-adenovirus neutralizing antibodies for three mice from each cohort were determined.
  • the mice which received two injections of Av3nLacZ without immunosuppression had relatively high antibody titers, while mice immunosuppressed with DSG and cyclophosphamide at the time of both " adenovira vector administrations had undetectable levels .
  • mice 1 and 4-6 The twelve mice which received two injections of Av3nLacZ (cohorts 1 and 4) , plus six naive mice (cohort 6) , each received 1 x 10 8 pfu of AvlH9F2 thirty-five days after the second Av3nLacZ administration. One week later, the mice were bled and plasma levels of human Factor IX were determined by ELISA.
  • Figure 22 is a graph of the results.

Abstract

Une méthode de thérapie génique chez un hôte consiste: (a) à administrer à cet hôte, conjointement, (i) un vecteur adénoviral comportant au moins une séquence d'ADN codant un agent thérapeutique, (ii), de la déoxyspergualine et, (iii), de la cyclophosphamide; (b) à interrompre l'administration de ces produits; et (c), à la reprendre au moins une fois. La répétition d'un traitement consistant dans l'administration d'un vecteur adénoviral, de déoxyspergualine ainsi que de cyclophosphamide se solde par une expression ininterrompue ou renforcée d'au moins une séquence d'ADN codant l'agent thérapeutique.
PCT/US1997/006199 1996-04-19 1997-04-15 Therapie genique reposant sur l'administration conjointe et repetee d'adenovirus et d'agents immunosuppresseurs WO1997039776A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU26688/97A AU2668897A (en) 1996-04-19 1997-04-15 Gene therapy involving concurrent and repeated administration of adenoviruses and immunosuppressive agents

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US63477496A 1996-04-19 1996-04-19
US08/634,774 1996-04-19

Publications (2)

Publication Number Publication Date
WO1997039776A1 WO1997039776A1 (fr) 1997-10-30
WO1997039776A9 true WO1997039776A9 (fr) 1998-01-29

Family

ID=24545153

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/006199 WO1997039776A1 (fr) 1996-04-19 1997-04-15 Therapie genique reposant sur l'administration conjointe et repetee d'adenovirus et d'agents immunosuppresseurs

Country Status (2)

Country Link
AU (1) AU2668897A (fr)
WO (1) WO1997039776A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19711803A1 (de) * 1997-03-21 1998-09-24 Hoechst Ag Verlängerung der Expression von transgenen Proteinen durch immunmodulierende Behandlung mit 15-Deoxyspergualin
WO1999058156A1 (fr) * 1998-05-12 1999-11-18 Baylor College Of Medicine Prevention du cancer par des methodes d'apport selectives
WO2000074688A1 (fr) * 1999-06-08 2000-12-14 The Children's Hospital Of Philadelphia Methodes permettant de prevenir la formation d'anticorps inhibiteurs dans le cadre d'une therapie genique
US20020014242A1 (en) 2000-07-31 2002-02-07 Abraham Scaria Use of rapamycin to inhibit immune response and induce tolerance to gene therapy vector and encoded transgene products
KR102627561B1 (ko) 2018-06-29 2024-01-24 우한 뉴로프스 바이오테크놀로지 리미티드 컴퍼니 레버 유전성 시신경병증의 치료를 위한 조성물 및 방법
EP3840785A4 (fr) 2018-08-20 2022-07-13 Wuhan Neurophth Biotechnology Limited Company Compositions et méthodes de traitement de la neuropathie optique héréditaire de leber
CN113025633B (zh) 2019-12-09 2024-08-27 武汉纽福斯生物科技有限公司 编码人nadh脱氢酶亚单位1蛋白的核酸及其应用
WO2024168252A2 (fr) * 2023-02-10 2024-08-15 Yale University Utilisation d'inhibiteurs de signalisation vegf-c pour inhiber des réponses immunitaires et augmenter l'efficacité de thérapie génique

Similar Documents

Publication Publication Date Title
WO1996012406A1 (fr) Therapie genique par administration concurrente et repetee d'adenovirus et d'agents immunodepresseurs
WO1996012406A9 (fr) Therapie genique par administration concurrente et repetee d'adenovirus et d'agents immunodepresseurs
EP0710288B1 (fr) Vecteurs adenoviraux pour le traitement de l'hemophilie
Christ et al. Gene therapy with recombinant adenovirus vectors: evaluation of the host immune response
EP0696206B1 (fr) Adenovirus contenant des proteines fibreuses modifiees
Tao et al. Sequestration of adenoviral vector by Kupffer cells leads to a nonlinear dose response of transduction in liver
Amalfitano et al. Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted
JP3565859B2 (ja) 改良されたアデノウイルスおよびその使用法
Brown et al. Helper-dependent adenoviral vectors mediate therapeutic factor VIII expression for several months with minimal accompanying toxicity in a canine model of severe hemophilia A
AU697022B2 (en) Methods and compositions for administering gene therapy vectors
Geest et al. Elimination of innate immune responses and liver inflammation by PEGylation of adenoviral vectors and methylprednisolone
Varnavski et al. Preexisting immunity to adenovirus in rhesus monkeys fails to prevent vector-induced toxicity
NZ330553A (en) Recombinase-mediated generation of adenoviral vectors where all adenoviral genes of all regions E1 to E4 and L1 to L5 of the adenoviral genome are eliminated
WO1998032842A1 (fr) Adenovirus comprenant des proteines d'hexon modifiees
NZ300387A (en) Adenoviral vector modified to reduce host immune and inflammatory responses and its use in gene therapy treatment
Seregin et al. Improving adenovirus based gene transfer: strategies to accomplish immune evasion
WO1997039776A9 (fr) Therapie genique reposant sur l'administration conjointe et repetee d'adenovirus et d'agents immunosuppresseurs
WO1997039776A1 (fr) Therapie genique reposant sur l'administration conjointe et repetee d'adenovirus et d'agents immunosuppresseurs
US6372208B1 (en) Method of reducing an immune response to a recombinant virus
US6251957B1 (en) Method of reducing an immune response to a recombinant virus
WO2005027711A2 (fr) Vecteurs d'adenovirus a capside modifiee et methodes d'utilisation
US6132989A (en) Methods and compositions for enhanced stability of non-adenoviral DNA
AU2002237910B2 (en) Method of enhancing delivery of a therapeutic nucleic acid
AU2002237910A1 (en) Method of enhancing delivery of a therapeutic nucleic acid
Podevin et al. In-vivo retroviral gene transfer to the liver is cancelled by an immune response against the corrected cells. Can it be avoided?
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载