Adeno-associated Virus (AAV) Capsid Chimeras with Enhanced Infectivity Reveal a Core Element in the AAV Genome Critical for both Cell Transduction and Capsid Assembly.

Lydia Viney,Modassir Choudhry,Tom Henley,Tilmann Bürckstümmer,Mario Mietzsch,Mavis Agbandje-Mckenna,Courtnee Eddington,Colin R Parrish

doi:10.1128/jvi.02023-20

Abstract

Adeno-associated viruses (AAV) have attracted significant attention in the field of gene and cell therapy due to highly effective delivery of therapeutic genes into human cells. The ability to generate recombinant AAV vectors compromised of unique or substituted protein sequences has led to the development of capsid variants with improved therapeutic properties. Seeking novel AAV vectors capable of enhanced transduction for therapeutic applications, we have developed a series of unique capsid variants termed AAV X-Vivo (AAV-XV) derived from chimeras of AAV12 VP1/2 sequences and the VP3 sequence of AAV6. These AAV variants showed enhanced infection of human primary T cells, hematopoietic stem cells, and neuronal cell lines over wildtype parental viruses, and superiority over AAV6 for genomic integration of DNA sequences by AAV alone or in combination with CRISPR gene editing. AAV-XV variants demonstrate transduction efficiency equivalent to AAV6 at multiplicities of infection 2 logs lower, enabling T cell engineering at low AAV doses. The protein coding sequence of these novel AAV chimeras revealed disruptions within the assembly-activating protein (AAP) which likely accounted for observed lower virus yield. A series of genome alterations, reverting the AAP sequence back to wildtype AAV6, had a negative impact on the enhanced transduction seen with AAV-VX, indicating overlapping functions within this sequence for both viral assembly and effective T cell transduction. Our findings show these AAV-XV variants are highly efficient at cell transduction at low dose and demonstrates the importance of the AAP coding region in both viral particle assembly and cell infection.IMPORTANCE A major hurdle to the therapeutic potential of AAV in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, ability to manufacture commercially viable titers of AAV to support this. By virtue of neutralizing antibodies against AAV that impede patient repeat-dosing, the dose of AAV for in vivo gene delivery has been high, which has resulted in unfortunate recent safety concerns and deaths in patients given higher-dose AAV gene therapy. We have generated new AAV variants possessing unique combinations of capsid proteins for gene and cell therapy applications termed AAV-XV, which have high levels of cell transduction and gene delivery at lower MOI. Furthermore, we demonstrate a novel finding, and an important consideration for recombinant AAV design, that a region of the AAV genome encoding the capsid viral protein and AAP is critical for both virus yield and the enhancement of infection/transduction.

Highlights

IMPORTANCE A major hurdle to the therapeutic potential of associated viruses (AAVs) in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, the ability to manufacture commercially viable titers of AAV to support this
A multiple sequence alignment between the selected serotypes and AAV6 showed conservation of functional regions that included a PLA2 motif [20], a calcium binding motif [21], and basic residue clusters that serve as nuclear localization sequences (NLS) [22], positioning of the latter was different for AAV5 (Fig. 2A)
Given the enhanced transduction of T cells and hematopoietic stem cells (HSCs) seen at a low multiplicities of infection (MOI) with capsid variants 5 and 5.3, we extended the analysis to other cell types to investigate their transduction efficiency

Summary

Introduction

IMPORTANCE A major hurdle to the therapeutic potential of AAV in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, the ability to manufacture commercially viable titers of AAV to support this. Despite broadly low innate immunogenicity, concerns over humoral immune responses against AAV capsids, observed in recent clinical trials, have been raised and are associated with high vector doses [3, 4] This in vivo limitation, as well as the high doses or multiplicities of infection (MOI) of virus required for sufficient cell transduction and the need to expand the repertoire of transducible tissue types addressable with AAV, is motivating further development of recombinant AAV (rAAV) technology. A targeted double-strand break (DSB) introduced by Cas at a specific location within the genome can be effectively repaired with an AAV template designed with homology to the target locus, via the pathway of homology-directed repair (HDR) [17] This AAV plus CRISPR combination approach has been effectively used by us and by others to perform genetic engineering of difficult-to-target cell types such as primary human T cells at levels of efficiency that are therapeutically relevant [18]. This enhanced transduction was further seen in other tissue types, including hematopoietic stem cells and neuronal cells

Methods

Results

Conclusion