Suppressing immunity to fight HIV infection

From the Jerome lab, Vaccine and Infectious Disease Division

Adeno-associated virus (AAV) vectors are harmless viruses that can be used to deliver gene-editing therapeutics. Some serotypes, such as AAV6, have the ability to deliver therapeutic transgenes to CD4+ T cells, the host cell for HIV, positioning AAV6 as a promising vehicle for anti-HIV therapeutics. However, as humans are a natural host for AAV, many people have pre-existing antibodies and T cells against AAV that can bind and neutralize AAV vectors and intercept their delivery of anti-HIV materials to the appropriate tissue sites. Dr. Dan Stone and Beth Kenkel from the Jerome lab, Vaccine, and Infectious Disease Division, suspected that immune suppression before treatment with AAV vectors could dampen pre-existing immunity to AAV and allow for delivery of vector contents, and tested this question in a recent pre-clinical study, published in Human Gene Therapy.

Although AAV6 can efficiently transduce CD4+ T cells in vitro, previous in vivo studies have revealed that AAV6 displays high tropism for many tissue and cell types, causing off-target sequestration and impaired delivery of vector cargo to CD4+ T cells when AAV6 is systemically administered. To mitigate this obstacle, the authors employed a previously described approach to retarget AAV tropism to CD4+ T cells, making this study “the first to demonstrate that AAV6 vectors can be used as a scaffold for vector engineering,” Dr. Stone said, through the use of “small targeting ligands on the AAV6 capsid such as direct ankyrin repeat proteins (DARPins).".

To determine if immune suppression by rapamycin, a drug that inhibits cytokine signaling and lymphocyte growth, could attenuate anti-AAV6 immunity and enhance the delivery of AAV6 vectors to CD4+ T cells, the authors used a non-human primate (NHP) model, which are also natural hosts of AAV. After confirming that the study animals possessed preexisting anti-AAV6 antibodies of similar titers but no anti-AAV6 T cells, the animals were treated with rapamycin and subsequently given a subcutaneous injection of control and CD4 retargeted AAV6 vectors. Blood draws and biopsies were performed to measure anti-AAV T cells and vector distribution. Additionally, to determine if subcutaneous rather than intravenous administration route could more efficiently deliver vectors to the lymphatics, where CD4+ T cells are abundant, an additional animal was given control AAV6 through the standard intravenous delivery route.

Analysis of biodistribution and gene transfer for AAV6 and CD4 retargeted AAV6-55.2 vectors in immune suppressed NHPs after intravenous or subcutaneous delivery. NHPs were immune suppressed with rapamycin for 1 or 2 weeks prior to AAV administration and for 2 weeks post AAV administration.
Analysis of biodistribution and gene transfer for AAV6 and CD4 retargeted AAV6-55.2 vectors in immune suppressed NHPs after intravenous or subcutaneous delivery. NHPs were immune suppressed with rapamycin for 1 or 2 weeks prior to AAV administration and for 2 weeks post AAV administration. Figure provided by Dr. Dan Stone.

PCR quantification revealed that vector distribution was widespread though most tissues, suggesting that retargeting the AAV6 vector did not decrease off-target sequestration. However, rapamycin treatment prevented anti-AAV T cell responses and allowed for muscle cell transduction by AAV6 vectors, demonstrating that immune suppression before and during AAV6 vector delivery can counteract neutralizing responses that interfere with AAV6 vector treatment. Importantly, blood work and histopathology revealed that treatment with rapamycin and engineered vectors was well tolerated and safe in NHP.

These results show that “even in the presence of pre-existing anti-AAV immunity, immune suppression enables efficient transduction of muscle by AAV6 vectors and stops the production of capsid or transgene-specific T cells in some animals,” Dr. Stone explained. “This is one of the first pre-clinical studies to analyze the safety and efficacy of an engineered AAV vector in NHPs. Our study demonstrates that even in the presence of pre-existing anti-AAV immunity, vector administration can be safe and efficient when subjects are immune suppressed with rapamycin,” he continued.

Although the primary aim of the study was successful, “detargeting and retargeting of AAV6 via surface display of a CD4-specific DARPin did not alter vector biodistribution relative to unmodified AAV6 vectors in NHPs, unlike in mice, so further studies are needed to understand these species-specific differences in vector sequestration within non-target organs,” Dr. Stone said. The authors are already pursuing these follow-up studies: “We recently performed a multiplexed study in immune suppressed NHPs using 11 different barcoded AAV vector capsids to determine which vectors perform best in different tissue types. This work was performed in collaboration with the Kiem lab at Fred Hutch and the Büning lab at Hannover Medical Center, and will help us identify the optimal vector capsid for delivery of anti-HIV therapeutics in an upcoming amfAR-funded study in SHIV infected NHPs,” Dr. Stone explained.

This work was supported by amfAR, the National Institute of Allergy and Infectious Diseases, and the University of Washington Center for AIDS Research.

UW/Fred Hutch Cancer Consortium members Hans-Peter Kiem and Keith Jerome contributed to this work.

Stone D, Kenkel EJ, Loprieno MA, Tanaka M, De Silva Feelixge HS, Kumar AJ, Stensland L, Obenza WM, Wangari S, Ahrens CY, Murnane RD, Peterson CW, Kiem HP, Huang ML, Aubert M, Hu SL, Jerome KR. 2020. Gene Transfer in Adeno-Associated Virus Seropositive Rhesus Macaques Following Rapamycin Treatment and Subcutaneous Delivery of AAV6, but Not Retargeted AAV6 Vectors. Human Gene Therapy. 2020 Nov 2. doi: 10.1089/hum.2020.113. Online ahead of print.