Bridging from Intramuscular to Limb Perfusion Delivery of rAAV: Optimization in a Non-human Primate Study

Abstract

Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development program to regional limb perfusion, comparing two methods previously established for gene therapy, peripheral venous limb perfusion (VLP) and an intra-arterial push and dwell (IAPD) using rAAV1 and rAAV8 in a non-human primate (rhesus macaque) study. The rhesus AAT transgene was used with a c-myc tag to enable quantification of transgene expression. 5 cohorts of animals were treated with rAAV1-IM, rAAV1-VLP, rAAV1-IAPD, rAAV8-VLP, and rAAV8-IAPD (n = 2–3), with a dose of 6 × 10 12 vg/kg. All methods were well tolerated clinically. Potency, as determined by serum levels of AAT, of rAAV1 by the VLP method was twice that observed with direct IM injection; 90 μg/mL with VLP versus 38 μg/mL with direct IM injection. There was an approximately 25-fold advantage in estimated vector genomes retained within the muscle tissue with VLP and a 5-fold improvement in the ratio of total vector genomes retained within muscle as compared with liver. The other methods were intermediate in the potency and retention of vector genomes. Examination of muscle enzyme (CK) levels indicated rAAV1-VLP to be equally safe as compared with IM injection, while the IAPD method showed significant CK elevation. Overall, rAAV1-VLP demonstrates higher potency per vector genome injected and a greater total vector retention within the muscle, as compared to IM injection, while enabling a much greater total dose to be delivered, with equivalent safety. These data provide the basis for continuation of the dose escalation of the rAAV1-AAT program in patients and bode well for rAAV-VLP as a platform for replacement of secreted proteins.

Muscle-Directed Delivery of an AAV1 Vector Leads to Capsid-Specific T Cell Exhaustion in Nonhuman Primates and Humans

“…The level of protein expression is respectively VLP > IM > IAPD ( Table 1 ). 39 As expected, all the animals seroconverted after rAAV administration, as illustrated by detection of anti-AAV1 neutralizing antibodies (NAbs) and immunoglobulin G (IgG) ( Table 2 ). In contrast to results obtained in the phase II AATD clinical trial, these animals showed sporadic and transient elevation in serum creatine kinase or liver transaminases (alanine aminotransferase [ALT]/aspartate aminotransferase [AST]).…”

“…The rhAAT-cMyc fusion protein expression was measured at the mRNA level by qRT-PCR and at the protein level in serum by a semiquantitative immunoblot over time. 39 Our results show that the protein expression was detected in all groups, with a higher protein expression in the VLP group: up to an average of 90 mg/mL at 60 days post-injection. The level of protein expression is respectively VLP > IM > IAPD ( Table 1).…”

“… 33 Three different methods of delivery were tested: IM (n = 3), VLP (n = 3), and IAPD (n = 2) ( Table 1 ). 39 As previously described by our group, 39 the procedures are well tolerated, and the vector biodistribution analysis showed a broader distribution when the vector is delivered by VLP compared to IM injection. To track protein expression and differentiate endogenous AAT from recombinant AAT, a c-Myc tag was added to the recombinant rhesus AAT (rhAAT) sequence.…”

“…60,61 In the case of a rAAV1 IM delivery, the vector stays concentrated at the site of injection, as illustrated by the vector biodistribution data: the same dose is administered via IM, VLP, or IAPD delivery, but the vector biodistribution is different, with a less concentrated and more diffuse distribution in the injected limb after VLP or IAPD delivery. [37][38][39][62][63][64] Tregs can also mediate T cell exhaustion, 65 but this does not seem to be the case here. Indeed, both cell types are detected early post-injection; more importantly, exhausted T cells are detected after VLP and IAPD delivery, whereas Tregs are not.…”

AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons

“…In comparison to transgenic approaches, like gene knock-out, conditional gene knock-out, or gene-editing by CRISPR/Cas9, the current experimental design is less time consuming as well as less prone to potential off-target effects or developmental failure. Furthermore, using rAAVs for cargo delivery to post-mitotic PHNs supports current ideas [ 43 , 44 , 51 ] to use viral transductions in basic and translational approaches.…”