Hemophilia A is a common hereditary bleeding disorder that is characterized by a deficiency of human blood coagulation factor VIII (hFVIII). Previous studies with adeno-associated viral (AAV) vectors identified two liver-specific promoter and enhancer combinations (E03.TTR and E12.A1AT) that drove high level expression of a codon-optimized, B-domain-deleted hFVIII transgene in a mouse model of the disease. This study further evaluated these enhancer/promoter combinations in cynomolgus macaques using two different AAV capsids (AAVrh10 and AAVhu37). Each of the four vector combinations was administered intravenously at a dose of 1.2 × 10 genome copy/kg into five macaques per group. Delivery of the hFVIII transgene via the AAVhu37 capsid resulted in a substantial increase in hFVIII expression compared to animals administered with AAVrh10 vectors. Two weeks after administration of E03.TTR packaged within the AAVhu37 capsid, average hFVIII expression was 20.2 ± 5.0% of normal, with one animal exhibiting peak expression of 37.1% of normal hFVIII levels. The majority of animals generated an anti-hFVIII antibody response by week 8-10 post vector delivery. However, two of the five macaques administered with AAVhu37.E03.TTR were free of a detectable antibody response for 30 weeks post vector administration. Overall, the study supports the continued development of AAV-based gene therapeutics for hemophilia A using the AAVhu37 capsid.

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Understanding the complexity of the long-lived HIV reservoir during antiretroviral therapy (ART) remains a considerable impediment in research towards a cure for HIV. To address this, we developed a single-cell strategy to precisely define the unperturbed peripheral blood HIV-infected memory CD4+ T cell reservoir from ART-treated people living with HIV (ART-PLWH) via the presence of integrated accessible proviral DNA in concert with epigenetic and cell surface protein profiling. We identified profound reservoir heterogeneity within and between ART-PLWH, characterized by new and known surface markers within total and individual memory CD4+ T cell subsets. We further uncovered new epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that suggest infected cells with accessible provirus, irrespective of reservoir distribution, are poised for reactivation during ART treatment. Together, our findings reveal the extensive inter- and intrapersonal cellular heterogeneity of the HIV reservoir, and establish an initial multiomic atlas to develop targeted reservoir elimination strategies.

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach.

Adeno-associated viral (AAV) vectors can target the liver, making them an attractive platform for gene therapy approaches that require the correction of hepatocytes. Crigler-Najjar syndrome is an autosomal recessive disorder of bilirubin metabolism that occurs when the liver's uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) enzyme activity is partially or completely absent. This syndrome is characterized by elevated bilirubin levels in the blood. An AAV8 vector was developed expressing a codon-optimized human version of UGT1A1 from a liver-specific promoter. High doses of the vector rescued neonatal lethality in newborn UGT1 knockout (KO) mice, which serve as a model of Crigler-Najjar syndrome, and significantly increased survival from 5 to 270 days. Newborn UGT1 KO mice treated with AAV had serum total bilirubin levels that were 5.7 times higher than the levels seen in heterozygous and wild-type mice, likely due to dilution of vector genome copies (GC) in the liver resulting from a proliferation of hepatocytes during growth of the animal. The elevation in serum total bilirubin levels in adult UGT1 KO mice depended on the AAV8 vector dose. At doses <10 GC/mouse, total bilirubin levels returned to those seen in phototherapy-rescued UGT1 KO mice. Mice injected with vector at 10 or 3 × 10 GC/mouse had sustained reduced total bilirubin levels throughout the duration of the study. When an AAV8 vector was re-administered in mice with elevated total bilirubin levels, serum total bilirubin levels decreased to wild-type levels (0.1-0.3 mg/dL) in mice that received a vector dose of 3 × 10 GC/kg. Therefore, a low-level and likely transient decrease in serum total bilirubin during the first days of life is necessary for rescuing the lethal phenotype present in the neonatal UGT1 KO mouse. Furthermore, it was possible to ablate the elevated total bilirubin levels in adult mice by re-administering an AAV8 vector.

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Systemically delivered adeno-associated viral (AAV) vectors are now in early-phase clinical trials for a variety of diseases. While there is a general consensus on inclusion and exclusion criteria for each of these trials, the conditions under which vectors are infused vary significantly. In this study, we evaluated the impact of intravenous infusion rate of AAV8 vector in cynomolgus macaques on transgene expression, vector clearance from the circulation, and potential activation of the innate immune system. The dose of AAV8 vector in terms of genome copies per kilogram body weight and its concentration were fixed, while the rate of infusion varied to deliver the entire dose over different time periods, including 1, 10, or 90 minutes. Analyses during the in-life phase of the experiment included sequential evaluation of whole blood for vector genomes and appearance of proinflammatory cytokines. Liver tissues were analyzed at the time of necropsy for enhanced green fluorescent protein (eGFP) expression and vector genomes. The data were remarkable with a relative absence of any statistically significant effect of infusion time on vector transduction, safety, and clearance. However, some interesting and unexpected trends did emerge.

Systemic delivery of adeno-associated viral (AAV) vectors has been evaluated for the treatment of several liver diseases, including homozygous familial hypercholesterolemia, ornithine transcarbamylase deficiency, and hemophilia. Here, we evaluated this approach for the treatment of Crigler-Najjar syndrome. We administered wild-type rhesus macaques with 1.0 × 1013 or 2.5 × 1013 genome copies/kg of an AAV serotype 8 vector expressing a codon-optimized version of human uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) from a liver-specific promoter. We extensively studied vector biodistribution, transgene expression, and immune responses following vector administration. All rhesus macaques survived until their scheduled necropsy at day 56 and showed no clinical abnormalities during the course of the study. Macaques administered with either vector dose developed a T cell response to the AAV capsid and/or transgene. We mapped the immunodominant epitope in the human UGT1A1 sequence, and we found no correlation between peripheral and tissue-resident lymphocyte responses. Upon further investigation, we characterized CD107a+, granzyme B+, CD4+, and CD8+ transgene-specific cellular responses that were restricted to tissue-resident T cells. This study highlights the importance of studying immune responses at the vector transduction site and the limited usefulness of blood as a surrogate to evaluate tissue-restricted T cell responses.