Readministration Of Vector Research Articles

340 Anti-CD20 attenuates lung humoral and cellular immune responses: implications for gene therapy in Cystic Fibrosis

OBJECTIVES/GOALS: Re-administration of inhaled gene therapies has the potential to overcome low correction efficiencies and limiting immune responses observed in previous trials of gene therapy for Cystic Fibrosis. We therefore tested the hypothesis that pre-treatment with a B-cell depleting α ± CD20 antibody would permit vector re-administration. METHODS/STUDY POPULATION: We first selected Adenoviral (Ad) vectors to study initially due to their well-known ability to elicit potent immune responses. Mice were dosed with a depleting α ± CD20 antibody or isotype control 2 days prior to delivery of Ad-Luc. 4 weeks later, mice were euthanized to assess the development of anti-vector immune responses. Flow cytometry and single-cell RNA sequencing were used to evaluate the development of lung-resident memory cells. Serum and airway antibody responses were assessed by ELISA. After 4 weeks, mice were dosed with Ad-LacZ and euthanized 3 days later to assess efficiency of second-round gene transfer by β-galactosidase activity assay. Similar methods were used in a pilot experiment with Adeno-associated virus vector (AAV), but with euthanasia 3 weeks after secondary gene transfer. RESULTS/ANTICIPATED RESULTS: Delivery of Ad vectors leads to the development of lung-resident memory B and T-cells. The depletion of B-cells prior to first-round vector delivery attenuated airway T-cell infiltration and serum IgG production, abrogated mucosal IgG and IgA production, and completely rescued secondary gene transfer. Genetically modified mouse models suggest secreted antibodies are critical in prevention of vector redosing. AAV vectors were found to be less immunogenic than Ad vectors, with only partial reduction of second-round gene transfer. However, anti-CD20 provided no benefit for AAV redelivery. DISCUSSION/SIGNIFICANCE: Mucosal humoral immunity is critical in preventing re-administration of Adenoviral vectors. Impairment of B-cell responses by α ± CD20 treatment prior to vector delivery allows re-administration and may help overcome low efficiencies of CF gene therapy. AAV vectors may be less susceptible to neutralization by pre-existing immunity.

B cell focused transient immune suppression protocol for efficient AAV readministration to the liver

Adeno associated virus (AAV) vectors are utilized for correcting multiple genetic disorders. Although the goal is to achieve life-long correction with a single vector administration, the ability to redose would enable extension of therapy in cases where initial gene transfer is insufficient to achieve a lasting cure, episomal vector forms are lost in growing organs of pediatric patients, or transgene expression is diminished over time. However, AAV typically induces potent and long-lasting neutralizing antibodies (NAb) against capsid that prevents re-administration. To prevent NAb formation in hepatic AAV8 gene transfer, we developed a transient B cell targeting protocol using a combination of monoclonal antibody therapy against CD20 (for B cell depletion) and BAFF (to slow B cell repopulation). Initiation of immunosuppression prior to (rather than at the time of) vector administration and prolonged anti-BAFF treatment prevented immune responses against the transgene product and abrogated prolonged IgM formation. As a result, vector re-administration after immune reconstitution was highly effective. Interestingly, re-administration before the immune system had fully recovered achieved further elevated levels of transgene expression. Finally, this immunosuppression protocol reduced Ig mediated AAV uptake by immune cell types with implications to reduce the risk of immunotoxicities in human gene therapy with AAV.

Long-Term Persistence of Antibodies to Adeno-Associated Viral Vectors Following Gene Therapy with scAAV8-LP1-Fixco

Background Gene therapy for hemophilia B (HB) consisting of a one-time, systemic, administration of adeno-associated viral (AAV) vectors encoding a functional Factor-IX gene is now approved and represents an important and long-awaited milestone. A rise in anti-AAV antibody titers follows systemic administration of AAV vectors. This does not have direct clinical consequences; however, persistence of neutralising anti-AAV antibodies (NAB) at high titers will preclude subsequent successful gene transfer with vector of the same serotype. This would be problematic, in the event that transgene expression should fall below therapeutic levels and repeat dosing might be needed. Furthermore, high titer NAB response to one serotype can cross-react with other serotypes thereby limiting the option of using alternative serotype AAV vectors. We report a longitudinal analysis of anti-AAV antibody response over a 10-year period in severe HB participants following systemic administration of scAAV2/8-LP1-hFIXco from the St Jude-UCL Phase I/II, gene therapy trial (AGT4HB ClinicalTrials.gov:NCT00979238). Methods Anti-AAV antibody levels were evaluated in the 10 trial participants following a bolus intravenous infusion of scAAV2/8-LP1-hFIXco at a dose of 2x10 11vg/kg (N=2), 6x10 11vg/kg (N=2), or 2x10 12vg/kg (N=6) between 2010 and 2012. Total anti-AAV antibodies (TAB) were measured by ELISA, and levels quantitated using a pooled IgG reference. NAB were quantitated by an in-vitro transduction inhibition assay (TIA) in which plasma samples were preincubated with AAV CMV NanoLuc (MOI: AAV3 1, AAV5 100 and 8 100) prior to evaluation of transduction of HEK 293T cells. Results were normalised to virus control and the 50% inhibition dilution (IC50) was calculated. For comparison, we assessed anti-AAV antibody titers in 38 normal human plasma samples using the same methodology. Results At the time of data cut-off (31 st Dec 2022) the median follow-up for the cohort was 10.7 years (range: 4-12 years) with follow-up in two participants (one mid dose and the other high dose) being limited to 4 years. A dose dependent AAV8 TAB response was observed with low dose participants having a mean of 84±101mg/ml at 1-year, mid dose 507 mg/ml and high dose 1650±603 mg/ml. This represented a >1400-fold increase from baseline, pre-AAV administration values. These TAB levels were significantly higher than levels following natural infection in 38 seropositive individuals not exposed to recombinant vector (normal range 0.31-34.02mg/ml). In the high dose cohort, TAB titer against AAV3 and AAV5 at 1 year after gene transfer were 145±167 mg/ml (natural infection reference range 0.31-29.61mg/ml) and 96±55 mg/ml (normal range 0.31-36.13mg/ml) respectively, consistent with cross-reactivity between AAV serotypes. There was a decline in TAB levels against all serotypes prior to reaching a plateau. At year 5 the high dose cohort was 928.9 ±556.9mg/ml for AAV8, 90.4±95.6 mg/ml for AAV3 and, 72.8 ±49.1 mg/ml for AAV5. AAV8 NAB titers had a similar kinetic with IC50 at 1 year 7261.5 ±9812 (low-dose), 15000 (mid-dose) and, 40204± 37823 (high dose). For comparison, AAV8 IC50 were significantly higher than levels following natural infection with wild-type AAV in seropositive individuals (range 5-747) (Mann Whitney p<0.0001). NABs levels declined to an IC50 level of 12907±8246 at 5 years for the high dose cohort, which was still 3000-fold higher than their baseline value (range 5-70.1) (Mann Whitney p<0.0001), and above the threshold for efficient gene transfer. The development of cross-reactive NAB levels was relatively modest and within the range observed in natural seropositive individuals. For the high dose cohort NAB to AAV3 at year 1 was 6536± 1869 (normal range 5- 13502), and for AAV5 at year 1 852± 596 (normal range 5-2143). Conclusion Following AAV gene therapy there is a robust dose dependent rise in anti-AAV 8 antibody levels that decline over time, but at 10 years are still significantly above the threshold for re-administration of vector of the same serotype. There was clear evidence of cross reactivity against AAV3 and 5. AAV5 NAB titres were lower than other serotypes declining to levels below a threshold required for efficient gene transfer in the low and mid dose cohort within 10 years. This suggests that, should FIX levels fall below therapeutic levels in this cohort, successful re-administration of an alternative vector may be possible.

Rescue of infant progressive familial intrahepatic cholestasis type 3 mice by repeated dosing of AAV gene therapy

Background & AimsGene therapy using recombinant adeno-associated virus (rAAV) vector carrying multidrug resistance protein 3 (MDR3) coding sequence (AAV8-MDR3) represents a potential curative treatment for progressive familial intrahepatic cholestasis type 3 (PFIC3), which presents in early childhood. However, patients with the severest form of PFIC3 should receive treatment early after detection to prevent irreversible hepatic fibrosis leading ultimately to liver transplantation or death. This represents a challenge for rAAV-based gene therapy because therapeutic efficacy is expected to wane as rAAV genomes are lost owing to hepatocyte division, and the formation of AAV-specific neutralising antibodies precludes re-administration. Here, we tested a strategy of vector re-administration in infant PFIC3 mice with careful evaluation of its oncogenicity – a particular concern surrounding rAAV treatment. MethodsAAV8-MDR3 was re-administered to infant Abcb4-/- mice 2 weeks after a first dose co-administered with tolerogenic nanoparticles carrying rapamycin (ImmTOR) given at 2 weeks of age. Eight months later, long-term therapeutic efficacy and safety were assessed with special attention paid to the potential oncogenicity of rAAV treatment. ResultsCo-administration with ImmTOR mitigated the formation of rAAV-specific neutralising antibodies and enabled an efficacious second administration of AAV8-MDR3, resulting in stable correction of the disease phenotype, including a restoration of bile phospholipid content and healthy liver function, as well as the prevention of liver fibrosis, hepatosplenomegaly, and gallstones. Furthermore, efficacious repeat rAAV administration prevented the appearance of liver malignancies in an animal model highly prone to developing hepatocellular carcinoma. ConclusionsThese outcomes provide strong evidence for rAAV redosing through co-administration with ImmTOR, as it resulted in a long-term therapeutic effect in a paediatric liver metabolic disorder, including the prevention of oncogenesis. Impact and implicationsRedosing of gene therapy for inborn hepatobiliary disorders may be essential as effect wanes during hepatocyte division and renewal, particularly in paediatric patients, but the approach may carry long-term risks of liver cancer. Viral vectors carrying a therapeutic gene exerted a durable cure of progressive familial intrahepatic cholestasis type 3 in infant mice and reduced the risk of liver cancer only following a second administration.

Immune profiling of adeno-associated virus response identifies B cell-specific targets that enable vector re-administration in mice.

Adeno-associated virus (AAV) vector-based gene therapies can be applied to a wide range of diseases. AAV expression can last for months to years, but vector re-administration may be necessary to achieve life-long treatment. Unfortunately, immune responses against these vectors are potentiated after the first administration, preventing the clinical use of repeated administration of AAVs. Reducing the immune response against AAVs while minimizing broad immunosuppression would improve gene delivery efficiency and long-term safety. In this study, we quantified the contributions of multiple immune system components of the anti-AAV response in mice. We identified B-cell-mediated immunity as a critical component preventing vector re-administration. Additionally, we found that IgG depletion alone was insufficient to enable re-administration, suggesting IgM antibodies play an important role in the immune response against AAV. Further, we found that AAV-mediated transduction is improved in µMT mice that lack functional IgM heavy chains and cannot form mature B-cells relative to wild-type mice. Combined, our results suggest that B-cells, including non-class switched B-cells, are a potential target for therapeutics enabling AAV re-administration. Our results also suggest that the µMT mice are a potentially useful experimental model for gene delivery studies since they allow repeated dosing for more efficient gene delivery from AAVs.

Overcoming the Challenges Imposed by Humoral Immunity to AAV Vectors to Achieve Safe and Efficient Gene Transfer in Seropositive Patients.

One of the major goals of in vivo gene transfer is to achieve long-term expression of therapeutic transgenes in terminally differentiated cells. The extensive clinical experience and the recent approval of Luxturna® (Spark Therapeutics, now Roche) and Zolgensma® (AveXis, now Novartis) place vectors derived from adeno-associated viruses (AAV) among the best options for gene transfer in multiple tissues. Despite these successes, limitations remain to the application of this therapeutic modality in a wider population. AAV was originally identified as a promising virus to derive gene therapy vectors because, despite infecting humans, it was not associated with any evident disease. Thee large proportion of AAV infections in the human population is now revealing as a limitation because after exposure to wild-type AAV, anti-AAV antibodies develops and may neutralize the vectors derived from the virus. Injection of AAV in humans is generally well-tolerated although the immune system can activate after the recognition of AAV vectors capsid and genome. The formation of high-titer neutralizing antibodies to AAV after the first injection precludes vector re-administration. Thus, both pre-existing and post-treatment humoral responses to AAV vectors greatly limit a wider application of this gene transfer modality. Different methods were suggested to overcome this limitation. The extensive preclinical data available and the large clinical experience in the control of AAV vectors immunogenicity are key to clinical translation and to demonstrate the safety and efficacy of these methods and ultimately bring a curative treatment to patients.

Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice.

Many inborn errors of metabolism require life-long treatments and, in severe conditions involving the liver, organ transplantation remains the only curative treatment. Non-integrative AAV-mediated gene therapy has shown efficacy in adult patients. However, treatment in pediatric or juvenile settings, or in conditions associated with hepatocyte proliferation, may result in rapid loss of episomal viral DNA and thus therapeutic efficacy. Re-administration of the therapeutic vector later in time may not be possible due to the presence of anti-AAV neutralizing antibodies. We have previously shown the permanent rescue of the neonatal lethality of a Crigler-Najjar mouse model by applying an integrative gene-therapy based approach. Here, we targeted the human coagulation factor IX (hFIX) cDNA into a hemophilia B mouse model. Two AAV8 vectors were used: a promoterless vector with two arms of homology for the albumin locus, and a vector carrying the CRISPR/SaCas9 and the sgRNA. Treatment of neonatal P2 wild-type mice resulted in supraphysiological levels of hFIX being stable 10 months after dosing. A single injection of the AAV vectors into neonatal FIX KO mice also resulted in the stable expression of above-normal levels of hFIX, reaching up to 150% of the human levels. Mice subjected to tail clip analysis showed a clotting capacity comparable to wild-type animals, thus demonstrating the rescue of the disease phenotype. Immunohistological analysis revealed clusters of hFIX-positive hepatocytes. When we tested the approach in adult FIX KO mice, we detected hFIX in plasma by ELISA and in the liver by western blot. However, the hFIX levels were not sufficient to significantly ameliorate the bleeding phenotype upon tail clip assay. Experiments conducted using a AAV donor vectors containing the eGFP or the hFIX cDNAs showed a higher recombination rate in P2 mice compared to adult animals. With this study, we demonstrate an alternative gene targeting strategy exploiting the use of the CRISPR/SaCas9 platform that can be potentially applied in the treatment of pediatric patients suffering from hemophilia, also supporting its application to other liver monogenic diseases. For the treatment of adult patients, further studies for the improvement of targeting efficiency are still required.

Tocilizumab As a Novel Immunomodulatory Regimen for Hemophilia Gene Therapy

Hemophilia is an X-linked bleeding disorder typically resulting in deficiency of factor VIII (FVIII) or factor IX (FIX) due to mutations in the F8 or F9 genes, respectively. Data from clinical trials have shown that the investigational delivery of functional F8 or F9 gene by recombinant adeno-associated virus (AAV) to hepatocytes can substantially eliminate the need for infusions of clotting factor in hemophilia patients. Despite major advances, the durability and redosing of these investigational gene therapies have been limited by the host immune response against the AAV capsid (Verdera et al. Mol Ther 2020). Currently, an oral corticosteroid, prednisone, is commonly used to prevent cytotoxic T cells from killing AAV-transduced hepatocytes and to sustain the production of transgenic clotting factor. However, in some instances, transgene expression was lost despite prednisone administration and prolonged use of prednisone can be associated with adverse effects. Previously, we demonstrated that the anti-capsid humoral response depends on interleukin 6 (IL-6) secretion from human monocyte-related dendritic cells (Kuranda et al. JCI 2018). IL-6 signaling in response to AAV was also observed in human non-parenchymal liver cells in vitro, animal gene transfer models and AAV-based gene therapy trial for hemophilia B (Konkle et al. Blood 2021).Here, we investigated the effects of the IL-6 signaling blockade as a possible targeted approach to modulate AAV vector immunogenicity in hemophilia gene therapy using a non-human primate (NHP) model. Spk100 AAV capsid was used to deliver the human F9 gene. To prevent IL-6 signaling, we used a monoclonal antibody, tocilizumab (TCZ), which blocks the IL-6 receptor. TCZ is currently approved for use in several forms of arthritis and cytokine release syndrome. Ten male cynomolgus monkeys received an intravenous injection of Spk100-hFIX vector (4x10 12 vg/kg) and 5 of those animals received a single dose of TCZ (8 mg/kg) the day prior to vector administration and were monitored for 13 weeks.As assessed by an array of clinical and anatomic pathology parameters, the investigational use of gene therapy combined with prophylactic TCZ administration was safe and well-tolerated. TCZ did not interfere with vector biodistribution, liver transduction or the transgenic FIX production. Spk100-hFIX alone modestly increased IL-6 secretion from NHP peripheral blood mononuclear cells (PBMC) in vitro but, following the vector infusion in animals, plasma IL-6 levels did not change significantly. Overall, cytokine secretion in response to Spk100 capsid and hFIX protein was lower in PBMCs isolated from TCZ-treated animals compared to cells obtained from control animals. As expected, animals administered Spk100-hFIX alone developed anti-capsid antibodies. The administration of TCZ was associated with a lower level of anti-Spk100 IgM, IgG and neutralizing antibodies (NAb). While the blockade of IL-6 signaling was effective for about for 14 days post-vector infusion, the lower level of anti-Spk100 antibodies persisted for the entire study duration. In the TCZ group, 4 out of 5 animals had NAb titers equal to or below 1:10, theoretically compatible with vector readministration. In contrast, 4 out of 5 animals in the control group developed high titer NAbs following vector infusion. Importantly, TCZ reduced the detection of the capsid-specific TNFα-positive T cells that were observed in animals after vector infusion. Finally, livers from sacrificed animals were used to prepare liver non-parenchymal cells for ex vivo phenotyping. Compared to the control animals, liver-resident immune cells from the TCZ-treated group had increased basal IL-10 secretion, while liver sinusoidal endothelial cells (albumin-CD45-CD31+CD146+MHCII+) had lower surface levels of MHC class II molecules, suggesting an anti-inflammatory milieu in the TCZ-treated livers.Our results show that short-term prophylactic blockade of IL-6 signaling was safely used in NHPs and has the potential to reduce immune responses commonly observed post AAV vector infusion. These results support the continued investigation of tocilizumab as a targeted immunomodulatory regimen in liver gene therapy for hemophilia. DisclosuresKuranda: Spark Therapeutics: Current Employment. Weisshaar: Spark Therapeutics: Current Employment. Chen: Spark Therapeutics: Ended employment in the past 24 months. Smith: Spark Therapeutics: Current Employment. Beck: Spark Therapeutics: Current Employment. Kahle: Spark Therapeutics: Current Employment. Mingozzi: Spark Therapeutics: Current Employment.

Why is AAV FVIII gene therapy not approved by the US Food and Drug Administration yet?

The prospect of a clinical strategy using an adeno-associated virus (AAV) vector for expression of therapeutic levels of factor VIII (FVIII) has been highly desirable. This was initially anticipated by promising data from clinical studies on AAV5-FVIII in men with severe hemophilia A. However, long-term follow-up showed a unique efficacy concern on the sustainability and durability derived from a continuous decline in the FVIII transgene levels starting 1 year after vector injection through year 5. Additional follow-up of early-phase studies and outcomes of an ongoing phase 3 study will likely provide evidence on the feasibility of this approach. Here, the potential underlying mechanisms of the FVIII declining levels, together with the revision of several unique early and late onset findings, are discussed. The lack of long-term preclinical studies in large animal models prevents the firm conclusion that FVIII levels decline was unexpected. It is possible that the combination of vector manufacturing platform and dose, accompanied with ectopic expression of supraphysiologic levels of FVIII at short-term follow-up, may all contribute to the sustainability and durability of the transgene levels. Notably, vector readministration to further improve the FVIII levels is not feasible at this time. Thus, the need of a one-and-done AAV strategy to achieve sustain FVIII levels of expression is sine qua non to impact favorably the disease phenotype.

IgG-cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies.

Neutralizing antibodies to adeno-associated virus (AAV) vectors are highly prevalent in humans1,2, and block liver transduction3-5 and vector readministration6; thus, they represent a major limitation to in vivo gene therapy. Strategies aimed at overcoming anti-AAV antibodies are being studied7, which often involve immunosuppression and are not efficient in removing pre-existing antibodies. Imlifidase (IdeS) is an endopeptidase able to degrade circulating IgG that is currently being tested in transplant patients8. Here, we studied if IdeS could eliminate anti-AAV antibodies in the context of gene therapy. We showed efficient cleavage of pooled human IgG (intravenous Ig) in vitro upon endopeptidase treatment. In mice passively immunized with intravenous Ig, IdeS administration decreased anti-AAV antibodies and enabled efficient liver gene transfer. The approach was scaled up to nonhuman primates, a natural host for wild-type AAV. IdeS treatment before AAV vector infusion was safe and resulted in enhanced liver transduction, even in the setting of vector readministration. Finally, IdeS reduced anti-AAV antibody levels from human plasma samples in vitro, including plasma from prospective gene therapy trial participants. These results provide a potential solution to overcome pre-existing antibodies to AAV-based gene therapy.

Tracking Adeno-Associated Virus Capsid Evolution by High-Throughput Sequencing.

Despite early successes using recombinant adeno-associated virus (rAAV) vectors in clinical gene therapy trials, limitations remain making additional advancements a necessity. Some of the challenges include variable levels of pre-existing neutralizing antibodies and poor transduction in specific target tissues and/or diseases. In addition, readministration of an rAAV vector is in general not possible due to the immune response against the capsid. Recombinant adeno-associated virus (AAV) vectors with novel capsids can be isolated in nature or developed through different directed evolution strategies. However, in most cases, the process of AAV selection is not well understood and new strategies are required to define the best parameters to develop more efficient and functional rAAV capsids. Therefore, the use of barcoding for AAV capsid libraries, which can be screened by high-throughput sequencing, provides a powerful tool to track AAV capsid evolution and potentially improve AAV capsid library screens. In this study, we examined how different parameters affect the screen of two different AAV libraries in two human cell types. We uncovered new and unexpected insights in how to maximize the likelihood of obtaining AAV variants with the desired properties. The major findings of the study are the following. (1) Inclusion of helper-virus for AAV replication can selectively propagate variants that can replicate to higher titers, but are not necessarily better at transduction. (2) Competition between AAVs with specific capsids can take place in cells that have been infected with different AAVs. (3) The use of low multiplicity of infections for infection results in more variation between screens and is not optimal at selecting the most desired capsids. (4) Using multiple rounds of selection can be counterproductive. We conclude that each of these parameters should be taken into consideration when screening AAV libraries for enhanced properties of interest.

A Novel AAV-mediated Gene Delivery System Corrects CFTR Function in Pigs.

Cystic fibrosis is an autosomal-recessive disease that is caused by a mutant CFTR (cystic fibrosis transmembrane conductance regulator) gene and is characterized by chronic bacterial lung infections and inflammation. Complementation with functional CFTR normalizes anion transport across the airway surface. Adeno-associated virus (AAV) is a useful vector for gene therapy because of its low immunogenicity and ability to persist for months to years. However, because its episomal expression may decrease after cell division, readministration of the AAV vector may be required. To overcome this, we designed an integrating AAV-based CFTR-expressing vector, termed piggyBac (PB)/AAV, carrying CFTR flanked by the terminal repeats of the piggyBac transposon. With codelivery of the piggyBac transposase, PB/AAV can integrate into the host genome. Because of the packaging constraints of AAV, careful consideration was required to ensure that the vector would package and express its CFTR cDNA cargo. In this short-term study, PB/AAV-CFTR was aerosolized to the airways of CF pigs in the absence of the transposase. Two weeks later, transepithelial Cl- current was restored in freshly excised tracheal and bronchial tissue. Additionally, we observed an increase in tracheal airway surface liquid pH and bacterial killing in comparison with untreated CF pigs. Airway surface liquid from primary airway cells cultured from treated CF pigs exhibited increased pH correlating with decreased viscosity. Together, these results show that complementing CFTR in CF pigs with PB/AAV rescues the anion transport defect in a large-animal CF model. Delivery of this integrating viral vector system to airway progenitor cells could lead to persistent, life-long expression in vivo.

Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration

Gene therapy mediated by recombinant adeno-associated virus (AAV) vectors is a promising treatment for systemic monogenic diseases. However, vector immunogenicity represents a major limitation to gene transfer with AAV vectors, particularly for vector re-administration. Here, we demonstrate that synthetic vaccine particles encapsulating rapamycin (SVP[Rapa]), co-administered with AAV vectors, prevents the induction of anti-capsid humoral and cell-mediated responses. This allows successful vector re-administration in mice and nonhuman primates. SVP[Rapa] dosed with AAV vectors reduces B and T cell activation in an antigen-selective manner, inhibits CD8+ T cell infiltration in the liver, and efficiently blocks memory T cell responses. SVP[Rapa] immunomodulatory effects can be transferred from treated to naive mice by adoptive transfer of splenocytes, and is inhibited by depletion of CD25+ T cells, suggesting a role for regulatory T cells. Co-administration of SVP[Rapa] with AAV vector represents a powerful strategy to modulate vector immunogenicity and enable effective vector re-administration.

AAV-mediated delivery of optogenetic constructs to the macaque brain triggers humoral immune responses.

Gene delivery to the primate central nervous system via recombinant adeno-associated viral vectors (AAV) allows neurophysiologists to control and observe neural activity precisely. A current limitation of this approach is variability in vector transduction efficiency. Low levels of transduction can foil experimental manipulations, prompting vector readministration. The ability to make multiple vector injections into the same animal, even in cases where successful vector transduction has already been achieved, is also desirable. However, vector readministration has consequences for humoral immunity and gene delivery that depend on vector dosage and route of administration in complex ways. As part of optogenetic experiments in rhesus monkeys, we analyzed blood sera collected before and after AAV injections into the brain and quantified neutralizing antibodies to AAV using an in vitro assay. We found that injections of AAV1 and AAV9 vectors elevated neutralizing antibody titers consistently. These immune responses were specific to the serotype injected and were long lasting. These results demonstrate that optogenetic manipulations in monkeys trigger immune responses to AAV capsids, suggesting that vector readministration may have a higher likelihood of success by avoiding serotypes injected previously.NEW & NOTEWORTHY Adeno-associated viral vector (AAV)-mediated gene delivery is a valuable tool for neurophysiology, but variability in transduction efficiency remains a bottleneck for experimental success. Repeated vector injections can help overcome this limitation but affect humoral immune state and transgene expression in ways that are poorly understood. We show that AAV vector injections into the primate central nervous system trigger long-lasting and serotype-specific immune responses, raising the possibility that switching serotypes may promote successful vector readministration.

Strategies for Selection of AAV Vectors for Administration to Liver: Studies in Nonhuman Primates

Vectors based on adeno-associated virus (AAV) have demonstrated early promise in clinical trials, including published reports in patients with hemophilia B where therapeutic levels of factor IX have been achieved using AAV serotype 8, a member of the clade E family. With the success of these hemophilia B clinical trials utilizing AAV8, numerous alternative AAV capsid types that target the liver have begun to enter clinical testing across multiple disease indications. Specific properties of the various AAV capsids should be taken into account such as overall efficiency of hepatocyte gene transfer, differential gene transfer across the porto-central axis within the liver, durability of gene expression and the potential for vector re-administration. In this study, we evaluated these aspects of AAV-directed liver gene transfer in rhesus macaques across various capsid serotypes. Animals were injected with vectors expressing the secreted reporter gene rhesus bhCG and produced using different capsids [AAV5, AAV3b and two clade E vectors (AAVrh10 and AAV8)]. Nonhuman primates (NHPs) injected with clade E vectors expressing bhCG were administered 3 months later with AAV5 or AAV3b vectors expressing rhesus derived AFP. The key findings were: 1) in naïve animals, clade E vectors demonstrated the highest levels of periportal gene transfer with AAV5 vectors having the lowest levels of periportal gene transfer; 2) AAVrh10 and AAV5 elicited higher levels of neutralizing antibodies (NAb) than AAV8 and AAV3b; 3) significant animal-to-animal variation in transgene expression was noted with AAV3b in seronegative animals; and 4) within the short time frame tested NAb elicited from AAVrh10 appears to have inhibited subsequent in vivo transduction with the serologically distinct AAV3b serotype; prior exposure to AAV8 did not interfere with AAV3b transduction. These studies highlight the influence that capsids can play in efficiency and immunogenicity of AAV vectors for liver gene therapy. DisclosuresWilson:Dimension Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Solid Gene Therapy: Consultancy, Membership on an entity’s Board of Directors or advisory committees; REGENXBIO: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Kattenhorn:Dimension Therapeutics: Employment. Wadsworth:Dimension Therapeutics: Employment, Equity Ownership.

77. Antigen-Specific Modulation of Capsid Immunogenicity with Tolerogenic Nanoparticles Results in Successful AAV Vector Readministration

Gene transfer approaches based on the adeno-associated virus (AAV) vector platform have shown great therapeutic potential in both preclinical studies and clinical trials. Neutralizing immune responses to AAV, however, are an important limitation to the use of AAV vectors as therapeutic tools, as even low-titer anti-capsid neutralizing antibodies (NAb) can lead to vector clearance and lack of efficacy. In particular, anti-AAV NAbs develop at high titers following vector administration and persist for several years after AAV vector administration, making vector re-administration hard if not impossible. Here we tested a novel strategy to modulate immune responses directed against AAV vectors based on the co-administration of biodegradable tolerogenic poly(lactic acid co-glycolytic acid) (PLGA) nanoparticles (tNP) containing rapamycin at the time of vector administration. C57BL/6 mice (n=5/group) received an AAV8 vector encoding for luciferase (AAV8-Luc) at a dose of 4×1012 vg/kg injected intravenously alone, or formulated with empty PLGA nanoparticles (NP), or formulated with tNP containing 100 µg of rapamycin. Three weeks after treatment, anti-AAV8 antibodies were measured and animals received a second intravenous infusion with an AAV8 vector encoding for human coagulation factor IX (AAV8-hFIX) at a dose of 4×1012 vg/kg formulated with NP or tNP. An anti-AAV8 antibody ELISA and an in vitro neutralization assay was used to follow humoral immune responses to the vector. While no development of anti-AAV8 antibodies was observed in the tNP-treated animals after the first and second vector administration, control groups developed robust humoral immune responses to the AAV8 capsid, which prevented vector readministration. Consequently, efficient hFIX transgene expression deriving from the second AAV8 vector administration was observed only when tNP were used, at levels identical to animals that received only a single administration of AAV8-hFIX. Lack of antibody formation against the AAV8 capsid in animals treated with tNP was also accompanied by a downregulation of both CD4+ and CD8+ T cell responses in the liver. A mild decrease in the frequency of CD4+ T cells in spleen, with no change in frequency of regulatory T cells, were also noted. In a separate set of experiments, we tested the antigen-specificity of the treatment of tNP with AAV8 administration. Mice (n=5/group) received 4×1012 vg/kg of an AAV8-luc vector with tNP followed by either challenge with hFIX protein in complete Freund's adjuvant, AAV5-hFIX vector intravenous injection, or AAV8-hFIX vector intravenous injection. All animals pretreated with AAV8-luc and tNP developed antibodies against the hFIX and the AAV5 antigens, while they anti-AAV8 antibody titers were significantly decreased. In conclusion, tNP administration together withAAV vector prevents anti-capsid immune responses in an antigen-specific manner and allows for AAV vector readministration, addressing one of the most important challenges of the in vivo gene transfer field.

764. Dosing and Re-Administration of Intravenous Lentiviral Vector for Liver-Directed Gene Transfer in Young Rhesus Monkeys and ADA-Deficient Mice

Liver-directed gene transfer with lentiviral vectors (LV) is a treatment modality being investigated for a number of genetic diseases. To study re-administration of LV, rhesus monkeys and Adenosine deaminase (ADA) knockout mice were repeatedly dosed with vesicular stomatitis virus (VSV) pseudotyped LV. In some studies the LV were modified with polyethylene glycol (PEG) since others showed that PEG modification of lysine residues of VSV increases LV half-life in serum and reduces compliment-mediated inactivation. ADA-deficiency is a defect in purine metabolism that results in severe combined immunodeficiency due to substrate accumulation. Unlike humans, ADA-/- mice do not survive to day 21 if not treated, however, when treated with intravenous ADA LV, high ADA expression in the liver was associated with prolonged survival and immune reconstitution. Liver vector copy number (VCN) was determined by qPCR. Anti-vector IgG concentration in plasma was determined by ELISA, using VSV LV as the capture antigen and a mouse anti-VSV IgG standard. Untreated animals have 10 µg/ml of reactive IgG. Direct competitive ELISA was used to determine the PEG concentration on PEG LV. ADA-/- mice treated with 5×10e10TU/kg of ADA LV at birth did not develop anti-vector antibodies (n=12) and showed 0.3±0.23 liver VCN whereas ADA-/- mice immune reconstituted with ERT and treated with 1.5×10e10 TU/kg at 4 months (m) produced 100-fold more anti-vector IgG (n=5; 6890±1143µg/ml; p=0.01) with no difference in VCN when adjusted for dose. Similar results were found in rhesus monkeys treated with 2.0×10e9 TU/kg of LV at 1 month postnatal age (n=2; 4030±10µg/ml) compared to rhesus monkeys treated at 4 m postnatal age (n=2; 17800±300µg/ml). In immune competent ADA+/- mice treated with ADA LV at birth followed by unmodified enhanced green fluorescent protein (eGFP) LV (n=3; 0.05 VCN; 1390µg/ml) or PEG modified eGFP LV (n=3; 0.05 VCN; 1330µg/ml) at 8 m, the liver VCN and the anti-vector responses were not different. However, if mice were treated with an unmodified eGFP LV or PEG modified eGFP LV at 4 m and an unmodified ADA LV at 8 m, anti-vector IgG concentration was increased 10-fold with the unmodified eGFP LV (n=3; 114666±666µg/ml) compared to the PEG modified eGFP LV (n=3; 9733±333µg/ml); however, the second LV was inactivated and no vector was detected from mice in either group. When ADA+/- mice were treated with an unmodified ADA LV at birth and again at 5 m (booster), followed by an unmodified (n=5) or PEG modified (n=5) eGFP LV at 8 m, the third injection was associated with acute toxicity and reduced probability of survival in both groups (40%; p=0.01). In immune competent rhesus monkeys dosed with an unmodified LV carrying a non-expressed transgene at 3 m of age and followed by an unmodified LV or a PEG modified LV carrying a different non-expressed transgene at 6 m, the second vector was not detected in either group and both the unmodified LV (n=2; 15600±1,200µg/ml) and the PEG-modified LV (n=2; 15500±900µg/ml) had similar anti-vector responses. In conclusion, LV dosing at birth is associated with reduced anti-vector responses compared to dosing at ~3 m of age. Repeat LV dosing can result in inactivation of a second vector administration that is associated with higher anti-vector IgG responses and a higher probability of acute toxicity when dosed months apart. PEG modification does not appear to protect the second dose from inactivation or prevent toxicity.

Unprecedented short-term in vivo protection in mice with a single immunization of a DNA GP vaccine against heterologous mouse-adapted Ebola virus challenge

Abstract The recent Ebola outbreak in West Africa is a reminder that correlates of protection against filovirus infection are still not well understood. DNA vectors are a serology-independent platform that allow for vector re-administration with minimal side-effects. We designed a new micro-consensus DNA vaccine that expresses a Zaire Ebolavirus (EBOV) glycoprotein (GP) based on 2002–2008 EBOV outbreak strains. This novel glycoprotein is 3% distant from the GP expressed by the 1976 EBOV Mayinga outbreak strain. The optimized GP DNA vaccine was administered in mice by intramuscular injection followed by electroporation (IM-EP) and elicited strong total IgG antibody and T cell responses. To assess protection from challenge, we administered the GP DNA vaccine or pVax1 control (40ug) to BALB/c mice (n=10/group) by IM-EP at days 0, -7, -14, or -28 before lethal challenge (1000LD50) with a mouse-adapted EBOV Mayinga strain. Importantly we observed 100% protection against the lethal heterologous challenge when the DNA vaccine was administered 28 days before challenge illustrating the potency of this vaccine in this model. More importantly, we observed 100% protection and 90% protection when the DNA vaccine was administered at days -14 and day -7, respectively. Most surprising was the observation of 40% survival in the group that received vaccine at Day 0 (2–3 hours) before challenge. Rapid, short-term protection has been previously observed with a VSV-ZEBOVGP viral vector vaccine but never before with a DNA vaccine. The data suggests that protection in mice following administration of a heterologous DNA vaccine can be afforded by mechanisms independent solely of antibody titers. Ongoing studies are underway in NHPs to investigate these findings.

324. Successful Repeated Delivery of Helper-Dependent Adenoviral Vector Toachieve Efficient Long-Term Human CFTR Expression in Mouse Airwaythrough Transient Immunosuppression

Cystic Fibrosis (CF) is a common life-threatening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel. As a chronic, lifelong disease, CF should be best treated with a continue level of CFTR expression. Pulmonary gene therapy may ultimately cure the CF lung disease. Thus, efficient long-term expression of the delivered CFTR gene in targeted cell types is essential to achieve this goal either by repeated application or with a long-duration expression system. Even though novel approaches to target airway progenitor/stem cells with gene editing may be more attractive, the efficiency for targeting specific progenitor cells will still be a challenge in airway delivery. Repeated administration of viral vectors may be required to increase the percentage of cells to be targeted and to boost long-term therapeutic effects. However, immune responses to viral proteins are the greatest barrier to repeated vector administration. Here, we demonstrated that repeated transduction of human CFTR gene in mouse airway can be successfully achieved with helper-dependent adenoviral (HD-Ad) vector through transient immunosuppression.We showed previously that cyclophosphamide significantly enhanced human CFTR expression in mice received HD-Ad vector readministration and sustained expression through inhibition of host immune reactions. In this study, we examined cyclophosphamide effects on expression of the human CFTR in multiple rounds of HD-Ad vector delivery to mouse airways. Four group mice were nasally delivered with an HD-Ad-K18-CFTR vector at 1.5×1010 vector particles per mouse at day 0, day 70 and day 120. Two groups were treated with cyclophosphamide 6 hours before and 4 day and 8 days after vector delivery each time (except the last round of vector delivery for mice sacrificed at day 123). The other two groups without treatment were used as controls. One treated group and one control group were sacrificed at day 123, and the others at day 153 for detecting transgene expression and immune reaction. We found that cyclophosphamide has significantly improved the expression of CFTR (4.1 times higher) compared to the control group as determined by real-time qPCR 3 days after last delivery. And the CFTR expression level was 3.1 times higher 30 days after last vector transduction. Cyclophosphamide also significantly reduced the levels of antiadenoviral and neutralizing antibodies in both BALF and serum as well as prevented leukocyte infiltration in lung tissues. This data suggested that efficient repeated transduction of human CFTR gene with HD-Ad vector can be achieved by transient immunopuression in mouse lung.

Slow AAV2 clearance from the brain of nonhuman primates and anti-capsid immune response.

Adeno-associated virus serotype 2 (AAV2) has previously been reported to be a slowly uncoating virus in peripheral tissues, but persistence of intact vector in primate brain has not been explored. Because some neurological gene therapies may require re-administration of the same vector to patients, it seems important to understand the optimal timeframe in which to consider such repeat intervention. Surprisingly, convection-enhanced delivery of AAV2 into the thalamus of nonhuman primates (NHPs) resulted in robust staining of neurons with A20 antibody that detected intact AAV2 particles at ∼1.5 months after infusion. However, by 2.5 months, no A20 staining was visible. These data confirmed earlier findings of persistence of intact AAV2 particles in ocular and hepatic tissues. In order to probe the potential consequences of this persistence, we infused AAV2-human aromatic L-amino acid decarboxylase into left and right thalamus of three NHPs, with a 3-month delay between infusions. During that interval, we immunized each animal subcutaneously with AAV2 virus-like particles (empty vector) in order to induce strong anti-capsid humoral immunity. Various high neutralizing antibody titers were achieved. The lowest titer animal showed infiltration of B lymphocytes and CD8(+) T cells into both the secondary and primary infusion sites. In the other two animals, extremely high titers resulted in no transduction of the second site and, therefore, no lymphocytic infiltration. However, such infiltration was prominent at the primary infusion site in each animal and was associated with overt neuronal loss and inflammation.