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 × 1012 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.

Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In α-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create a recipient strain (NSG-PiZ) for human hepatocyte xenotransplantation. Results indicate that NSG-PiZ recipients support heightened engraftment of normal human primary hepatocytes as compared with NSG recipients. This model can therefore be used to test hepatocyte cell therapies for AATD, but more broadly it serves as a simple, highly reproducible liver xenograft model. Finally, a promoterless adeno-associated virus (AAV) vector, expressing a wild-type AAT and a synthetic miRNA to silence the endogenous allele, was integrated into the albumin locus. This gene-editing approach leads to a selective advantage of edited hepatocytes, by silencing the mutant protein and augmenting normal AAT production, and improvement of the liver pathology.

A number of packaging systems are available for production of recombinant adeno-associated virus vectors (rAAVs). Among these, the use of a two-plasmid cotransfection system, in which Rep and Cap genes and Ad helper genes are on the same plasmid, has not been frequently employed for good manufacturing practices (GMP) production, even though it presents some practical advantages over the common three-plasmid (triple) transfection method. To confirm and expand the utility of the two-plasmid system, we generated GMP-compatible versions of this system and used those package reporter genes in multiple capsid variants in direct comparison with triple transfection. Vector yields, purity, and empty-to-full ratios were comparable between double and triple transfection methods for all capsid variants tested. We performed an in vivo side-by-side comparison of double and triple transfection vectors following both intravenous injection and intramuscular injection in mice. Expression and transduction were evaluated in muscle and liver 4 weeks after injection. Additional studies of bioactivity were conducted in vivo using packaged vectors carrying a variety of cargos, including the therapeutic transgene, microRNA, and single- or double-stranded vector. Results showed that cargos packaged using double transfection were equivalently bioactive to those packaged using a triple transfection system. In conclusion, these data suggest the utility of midrange (1E12-1E16) GMP-compatible packaging of adeno-associated virus (AAV) vectors for several AAV capsids.

α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

Evaluating the performance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays and clearly articulating the utility of selected antigens, isotypes, and thresholds is crucial to understanding the prevalence of infection within selected communities.

Recombinant adeno-associated virus serotype 3B (rAAV3B) can transduce cultured human liver cancer cells and primary human hepatocytes efficiently. Serine (S)- and threonine (T)-directed capsid modifications further augment its transduction efficiency. Systemically delivered capsid-optimized rAAV3B vectors can specifically target cancer cells in a human liver cancer xenograft model, suggesting their potential use for human liver-directed gene therapy. Here, we compared transduction efficiencies of AAV3B and AAV8 vectors in cultured primary human hepatocytes and cancer cells as well as in human and mouse hepatocytes in a human liver xenograft NSG-PiZ mouse model. We also examined the safety and transduction efficacy of wild-type (WT) and capsid-optimized rAAV3B in the livers of nonhuman primates (NHPs). Intravenously delivered S663V+T492V (ST)-modified self-complementary (sc) AAV3B-EGFP vectors led to liver-targeted robust enhanced green fluorescence protein (EGFP) expression in NHPs without apparent hepatotoxicity. Intravenous injections of both WT and ST-modified rAAV3B.ST-rhCG vectors also generated stable super-physiological levels of rhesus chorionic gonadotropin (rhCG) in NHPs. The vector genome predominantly targeted the liver. Clinical chemistry and histopathology examinations showed no apparent vector-related toxicity. Our studies should be important and informative for clinical development of optimized AAV3B vectors for human liver-directed gene therapy.

Immune responses to adeno-associated virus (AAV) capsids limit the therapeutic potential of AAV gene therapy. Herein, we model clinical immune responses by generating AAV capsid-specific chimeric antigen receptor (AAV-CAR) T cells. We then modulate immune responses to AAV capsid with AAV-CAR regulatory T cells (Tregs). AAV-CAR Tregs in vitro display phenotypical Treg surface marker expression, and functional suppression of effector T cell proliferation and cytotoxicity. In mouse models, AAV-CAR Tregs mediated continued transgene expression from an immunogenic capsid, despite antibody responses, produced immunosuppressive cytokines, and decreased tissue inflammation. AAV-CAR Tregs are also able to bystander suppress immune responses to immunogenic transgenes similarly mediating continued transgene expression, producing immunosuppressive cytokines, and reducing tissue infiltration. Taken together, AAV-CAR T cells and AAV-CAR Tregs are directed and powerful immunosuppressive tools to model and modulate immune responses to AAV capsids and transgenes in the local environment.