In vivo engineering of lymphocytes after systemic exosome-associated AAV delivery

Abstract

Ex-vivo gene therapy using stem cells or t cells transduced by retroviral or lentiviral vectors has shown remarkable efficacy in the treatment of immunodeficiencies and cancer. However, the process is expensive, technically challenging, and not readily scalable to large patient populations, particularly in underdeveloped parts of the world. Direct in vivo gene therapy would avoid these issues, and such approaches with adeno-associated virus (AAV) vectors have been shown to be safe and efficacious in clinical trials for diseases affecting differentiated tissues such as the liver and CNS. However, the ability to transduce lymphocytes with AAV in vivo after systemic delivery has not been carefully explored. Here, we show that both standard and exosome-associated preparations of AAV8 vectors can effectively transduce a variety of immune cell populations including CD4 + T cells, CD8 + t cells, B cells, macrophages, and dendritic cells after systemic delivery in mice. We provide direct evidence of t cell transduction through the detection of AAV genomes and transgene mRnA, and show that intracellular and transmembrane proteins can be expressed. These findings establish the feasibility of AAV-mediated in vivo gene delivery to immune cells which will facilitate both basic and applied research towards the goal of direct in vivo gene immunotherapies. Gene therapy using ex-vivo transduction of patient-derived cells has revolutionized medicine, particularly in the case of readily accessible blood hematopoietic stem cells or lymphocytes to treat immunodeficiencies or cancer 1-3. However, the laborious workflow and infrastructure required makes these therapies expensive, time-consuming, and unscalable. Moreover, the manipulation of cells outside the body may introduce undesirable phenotypic changes 4. Administering vectors directly into patients would solve most of these problems, potentially expanding the reach of gene therapies to large patient populations anywhere in the world, but unfortunately, such therapies have not advanced due to the absence of a proven and effective delivery system. AAV vectors were thoroughly developed over the past 35 years and are now demonstrating remarkable, life-changing efficacy in clinical trials for several diseases, including recently FDA approved treatments for a form of hereditary blindness and spinal muscular atrophy 5-9. The majority of AAV-mediated gene therapies have focused on differentiated cells such as muscle 10,11 , neurons, astrocytes 12 , and liver 13. AAV has generally been considered inefficient at transducing T cells; however, a recent study revealed that CD3 + T cells are a natural reservoir for multiple wild type AAV serotypes 14. Here we demonstrate that AAV8 vectors can mediate transgene expression in multiple immune cell types after systemic delivery in mice, providing an important proof of concept for developing these vectors for use in in vivo therapies.

On the Relationship of Viral Particles and Extracellular Vesicles: Implications for Viral Vector Technology

“…In an AV vector system, association of VV to vesicles improved the efficacy of oncolytic virus therapy on tumors with low coxsackie-and-adenovirus (CAR) [ 34 ]. In AAV exosome, associated AAV8 vectors were demonstrated to efficiently transduce lymphocytes after systemic delivery [ 39 ].…”

“…Similar to the situation for AV, knowledge on the influence of EVs on wt AAV infections is scarce. Indeed, information on wt parvovirus relationships to EVs is mostly found in the context of research on vector systems [ 39 , 40 ]. For other groups of non-enveloped viruses, such as members of the families Picornaviridae , Reoviridae and Papillomaviridae , interactions with EV are well documented.…”

AAV-Containing Exosomes as a Novel Vector for Improved Gene Delivery to Lung Cancer Cells

“…As an innovative approach, AAV-associated exosomes have been reported to efficiently deliver genes to the central nervous system (Hudry et al, 2016;Volak et al, 2018), immune cells (Breuer et al, 2020), retina (Wassmer et al, 2017), cochlea (György et al, 2017), and liver cells (Meliani et al, 2017). Our study has shown great potential that AAVExo may enhance the existing gene therapies for cancer treatment.…”

Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8

“…Only recently, in vivo transduction of mouse lymphocytes with serotype-8-derived vectors was described. 43 Our data suggest that efficient binding to T cells via CD8 enables efficient gene delivery with AAV2 vectors. This stresses the crucial role of high-affinity targeting ligands displayed on the particle surface.…”