Etude des facteurs modulant le transfert de gène dans le muscle squelettique de primate à l'aide d'un AAV recombinant

L'efficacité du transfert de gène au moyen d'un AAVr dans le muscle de primate peut être inhibée par le développement d'une réponse immunitaire. Nous avons donc étudié chez le macaque la relation entre le mode d'administration, le sérotype d'AAVr, la biodistribution du vecteur et le système immunitaire de l'hôte. Nos résultats montrent que la voie intramusculaire, contrairement à l'infusion de membre isolé, est associée au développement d'évènements immunologiques délétères dirigés contre le produit du transgène et la capside du vecteur.NANTES-BU Médecine pharmacie (441092101) / SudocTOULOUSE-EN Vétérinaire (315552301) / SudocSudocFranceF

Etude des facteurs modulant le transfert de gène dans le muscle squelettique de primate à l'aide d un AAV recombinant

L'Adeno-Associated Virus recombinant (AAVr) est un outil thérapeutique performant car, lorsque le système immunitaire n'est pas stimulé, le transfert de gène après une seule administration intramusculaire (IM) se traduit par une expression permanente du transgène. Cependant, l'efficacité du transfert de gène dans le muscle de primate peut être inhibée par le développement d'une réponse immunitaire contre le vecteur et/ou le produit du transgène. Les vecteurs AAVr étant de plus en plus utilisés en essais cliniques, chercher les facteurs impliqués dans l'émergence de cette réponse immunitaire est nécessaire. Nous avons donc étudié chez le macaque la relation entre le mode d administration (IM ou infusion de membre isolé : RI ), le sérotype d AAVr (1 ou 8), la biodistribution du vecteur et le système immunitaire de l hôte. Nos résultats montrent que la biodistribution du vecteur est semblable après IM et RI, mais que la voie IM, contrairement à la RI, est associée au développement d évènements immunologiques dirigés contre le produit du transgène et la capside du vecteur. L immunosuppresseur LEA29Y seul n est pas suffisant pour empêcher l activation d une telle réponse immune. Ainsi l injection IM d un AAVr chez le primate est associée à une réponse immunitaire délétère, quel que soit le sérotype utilisé. Pour les futurs essais cliniques, il nous apparaît donc indispensable d administrer le vecteur AAVr par une voie moins immunogène. L'injection d AAVr par RI semble être une voie prometteuse : elle n'est pas associée au développement d une réponse immune chez le primate et permet l expression à long terme du transgène, du moins dans le modèle développé dans notre laboratoire.Recombinant Adeno-Associated Virus (rAAV) are a powerful therapeutic tool, because when the immune system is not stimulated, gene transfer after a single intramuscular (IM) administration results in sustained transgene expression (> 6 years). However, the efficiency of gene transfer in nonhuman primates is inhibited by the development of an immune response against vector capsid and/or transgene product. AAV vectors being increasingly used in gene therapy clinical trials, looking for factors involved in the development of a such immune response is necessary. We therefore studied in cynomolgus macaques the relationship between the mode of delivery (IM or isolated limb infusion: RI), the rAAV serotype (1 or 8), the vector biodistribution and the host immune system. Our results show that the biodistribution of the vector is similar after IM or RI. However, IM delivery, unlike isolated limb infusion, is associated with the development of immunological events directed against the transgene product and the rAAV capsid. The immunosuppressive molecule LEA29Y alone is not sufficient to inhibit this immune response. Our conclusion is that IM injection of rAAV in primate is associated with a deleterious immune response, regardless of the serotype. For further clinical trials, it is therefore essential to administer AAV vectors via a less immunogenic mode of delivery than IM. Isolated limb infusion delivery seems to be a promising route, since it is not associated with the trigger of an immune response in the primate, and allows long-term transgene expression, at least in our model developed in our laboratory.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates

Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Kru ̈ ppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed

5 years of successful inducible transgene expression following locoregional AAV delivery in nonhuman primates with no detectable immunity

International audienc

Adeno-Associated Virus Vector Genomes Persist as Episomal Chromatin in Primate Muscle▿

Recombinant adeno-associated virus (rAAV) vectors are capable of mediating long-term gene expression following administration to skeletal muscle. In rodent muscle, the vector genomes persist in the nucleus in concatemeric episomal forms. Here, we demonstrate with nonhuman primates that rAAV vectors integrate inefficiently into the chromosomes of myocytes and reside predominantly as episomal monomeric and concatemeric circles. The episomal rAAV genomes assimilate into chromatin with a typical nucleosomal pattern. The persistence of the vector genomes and gene expression for years in quiescent tissues suggests that a bona fide chromatin structure is important for episomal maintenance and transgene expression. These findings were obtained from primate muscles transduced with rAAV1 and rAAV8 vectors for up to 22 months after intramuscular delivery of 5 × 1012 viral genomes/kg. Because of this unique context, our data, which provide important insight into in situ vector biology, are highly relevant from a clinical standpoint

Lack of Immunotoxicity After Regional Intravenous (RI) Delivery of rAAV to Nonhuman Primate Skeletal Muscle

International audienceIn the absence of an immune response from the host, intramuscular (IM) injection of recombinant adeno-associated virus (rAAV) results in the permanent expression of the transgene from mouse to primate models. However, recent gene transfer studies into animal models and humans indicate that the risk of transgene and/or capsid-specific immune responses occurs and depends on multiple factors. Among these factors, the route of delivery is important, although poorly addressed in large animal models. Here, we compare the IM and the drug-free regional intravenous (RI) deliveries of rAAV in nonhuman primate (NHP) skeletal muscle monitoring the host immune response toward the transgene. We show that IM is consistently associated with immunotoxicity and the destruction of the genetically modified myofibers, whereas RI allows the stable expression of the transgene. This has important implications for the design of clinical trials for gene transfer in skeletal muscle

Gene transfer of human CD40Ig does not prevent rejection in a non-human primate kidney allotransplantation model

International audienceBACKGROUND: Blockade of costimulation signaling required for immune response, such as CD40/CD40L and CD28/B7, is a reasonable strategy to prevent rejection and in defined combinations may allow donor specific tolerance. Indeed, in rodents, costimulation blockade with CD28/B7 antagonists or with CD40Ig was able to induce regulatory T cells and transplant tolerance whereas in primates, anti-CD40 antibodies, anti-CD40L antibodies or CTLA4Ig, used as monotherapy, significantly delayed graft rejection.METHODS: Using an adeno-associated virus (AAV) vector mediated gene transfer of a human CD40Ig fusion protein (hCD40Ig) in primates, we evaluated the capacity of this costimulation blockade molecule interfering with CD40/CD40L signaling in prolonging kidney transplants in cynomolgus monkeys.RESULTS: This gene transfer strategy allowed for maintaining a plateau of hCD40Ig production within two months and avoided a high-scale production phase of this molecule. Although the hCD40Ig was able to bind efficiently to human and macaque CD40L and high (>200μg/ml) transgene expression was obtained, no effect on graft survival was observed. In addition, there was no inhibition of humoral response to vaccination. In vitro, hCD40Ig strongly increased mixed lymphocyte reaction, and when compared to the anti-CD40L antibody h5C8, was not as potent to induce complement-dependent cytotoxicity.CONCLUSION: These data suggest that CD40/CD40L blockade using a non-depleting CD40Ig fusion protein, a therapeutic strategy that showed efficacy in rodents, is not able to modulate the immune response in primates. These data highlight important biological differences between rodent and primate models to evaluate therapeutic strategies at the preclinical level

rAAV.TetR-KRAB/cmEpo in the macaque muscle results in the loss of Dox-mediated transgene regulation.

<p>(<b>A</b>) Vector design: the vector encodes the Cynomolgus macaque <i>Epo</i> (<i>cmEpo</i>) under the control of the doxycycline-inducible <i>TetO</i>-PGK promoter and the TetR-KRAB chimeric protein under the control of the CAG promoter. Arrows indicate the transcription start sites. ITR: inverted terminal repeat sequence of AAV2; pA (mEpo), SV40 polyadenylation signal; pA (TetR-KRAB), BGH polyadenylation signal. The two expression cassettes were cloned between AAV2 ITRs in the same orientation (<i>forward</i>). (<b>B</b>) Follow-up of hematocrit, reticulocyte and <i>serum</i> Epo levels in Mac 1, Mac 2 and Mac 3 injected <i>via</i> IM route with 8×10¹⁰ vg/kg of rAAV1-TetR-KRAB/<i>cmEpo</i> vector for over 8 months. rAAV injection and Dox administrations are indicated with arrows <b>Upper panel</b>: Hematocrit levels (%) in dark lines, and reticulocyte numbers (10⁹/L of blood) in gray lines. <b>Lower panel</b>: <i>Serum</i> Epo levels (mU/mL). (<b>C</b>) <b>Detection of anti-TetR IgG antibodies</b> (Ab (TetR)) by western-blot analysis before rAAV administration (day 0) and at 3.5, 5, 6 and 7 months post-injection. C+: positive control consisting of a commercial specific monoclonal antibody against TetR.</p

rAAV.TetR-KRAB/mEpo in the mouse muscle results in Dox-mediated transgene regulation.

<p>(<b>A</b>) Vector design: the vector encoded the murine <i>Epo</i> (<i>mEpo</i>) under the control of a <i>TetO</i>.lCMVlg, <i>TetO</i>.CMVsh, or <i>TetO</i>.PGK promoter and the trans-inhibitor TetR-KRAB under the control of the CAG promoter. pA (<i>mEpo</i>), SV40 polyadenylation signal; pA (TetR-KRAB), BGH polyadenylation signal; ITR, inverted terminal repeat of AAV2. Arrows indicate the transcription start sites. All expression cassettes were cloned between AAV2 ITRs in the same orientation (<i>forward</i>). (<b>B</b>) Follow-up of mEpo levels in animal groups before and after Dox induction. Six to 8 mice were injected IM in the <i>tibialis</i> anterior muscle with a rAAV1 vector harboring either <i>TetO</i>-CMVlg (n = 8), <i>TetO</i>-CMVsh (n = 6) or <i>TetO</i>-PGK (n = 8) promoters at a dose of 3×10⁹ total viral genomes (vg) in a volume of 30 µL. A rAAV1 vector carrying an rtTA-<i>mEpo</i> cassette (<i>forward</i> orientation) was injected at the same dose and volume in the control group (n = 8). Epo levels in the absence and in the presence of Dox are presented as the mean + SD <i>per</i> group in pg/ml. Murine Epo baseline (dotted line) was determined as the mean Epo level (115 pg/ml) in non-injected mice (n = 83). Mann-Whitney statistical test was performed between (-Dox) and (+Dox) conditions for each experimental group. ***: p<0.001, **: p<0.01, *p<0.05.</p

Regulation of d2GFP expression in the rat retina using the TetR-KRAB system over a time period of 48 weeks.

<p>Expression of d2GFP was induced several times with Dox for over 48 weeks to evaluate the long-term regulation and functionality of the regulatory system after subretinal injection of (<b>A</b>) rAAV5.d2GFP.KRAB <i>forward</i> and (<b>B</b>) rAAV5.d2GFP.KRAB <i>opposite</i> vectors. Images of fundus fluorescence were taken 14 days after starting the administration of Dox (+Dox) or the withdrawal of Dox (-Dox) at 15, 20 and 38 weeks post-injection.</p