The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner

Abstract

Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species. In our study, we use a primate specific capsid, AAV-LK03, to demonstrate that the limitation of this capsid towards transduction of mouse cells is unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enables efficient transduction and the accumulation of active-related epigenetic marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in driving the epigenetic status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA transduction between vector and host species and ultimately lead to rational selection of AAV capsids for use in humans.

Natural Adeno-Associated Virus Serotypes and Engineered Adeno-Associated Virus Capsid Variants: Tropism Differences and Mechanistic Insights

“…Importantly, the AAV genome interacts with histones to form unique extrachromosomal chromatin structures [ 188 , 189 ] that are important both for in vivo episome persistence and transcriptional regulation [ 190 ]. Transcriptional regulation takes place by means of the HUSH complex and the DNA-binding protein NP220 via the epigenetic regulation of the bound histones [ 191 ] and can be influenced by the capsid and host species [ 191 , 192 ]. In rare cases, the AAV genome can also integrate into the host DNA at a very low frequency [ 87 , 89 ].…”

“…AAV-LK03 is a chimera of AAV3B, AAV1, 2, 4, 6, 8, and 9 engineered by DNA shuffling and screened in mice partially repopulated with primary human hepatocytes [ 286 ]. Interestingly, this variant showed efficient transgene expression in human but not mouse hepatoma cell lines, despite the detection of a similar number of vector genomes [ 192 , 286 ]. In addition, it transduced the hepatocytes of humanized mice (10-fold higher than AAV8) but not those of non-humanized controls, and it exhibited only the transduction of human hepatic tumors but not mouse hepatocytes in a hepatocellular carcinoma xenograft model [ 286 ].…”

“…A good example is AAV-LK03, a chimeric variant which shows a high transduction of human hepatocytes but no transduction of mouse hepatocytes [ 286 ] and exhibits a slower onset of transgene expression compared to AAV8. Interestingly, it was recently discovered that this variant is not defective in the mouse cell receptor binding, entry, uncoating, nuclear accumulation, or episome formation of vector genomes but rather shows a lack of histone modifications (H3K4me3 and H3K27ac) in the viral genome related to active transcription in mouse cells despite proper nucleosome assembly [ 192 ]. In contrast, the amount of repressive histone modifications (H3K9me3 and H3K27me3) is comparable to other capsid controls.…”

Adeno-Associated Viral Tools to Trace Neural Development and Connectivity Across Amphibians

“…The transient detection of AAV DNA/RNA ( Figure 3H ), but not GFP protein ( Figure 3I ), in radial glia indicates that viral genomes are either diluted rapidly during cell proliferation, or that AAV payload transcription or translation are impaired specifically in this cell type 89 . To distinguish between these two possibilities, we performed intracerebroventricular AAV injections in post-metamorphic juveniles (stage 56) 65 , in which the majority of radial glia cells have already transitioned to a quiescent state 12 .…”

“…This could be due to the translational repression of viral mRNA in these glial types. Alternatively, the AAV genome might be epigenetically silenced in radial glia 89 , or its translocation to the nucleus might be prevented, followed by degradation of the viral DNA, as is suggested for mouse microglia 120 . Interestingly, radial glia and EGCs are the glial cell types most closely related to mammalian astrocytes, which are transduced by AAV-PHP.eB with high efficiency 121 .…”