The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver

Abstract

IntroductionAdeno-associated viral (AAV) vectors are widely used for stable in vivo gene transfer to terminally differentiated or quiescent cells such as muscle fibers, hepatocytes, neurons, retinal cells, and others. These vectors, derived from a nonpathogenic, replicationdefective parvovirus with a small single-stranded (ss) DNA genome, have recently been successfully used in clinical gene transfer for inherited blindness and also show promise for other diseases. 1,2 Eight years ago, Zaiss et al 3 found that ssAAV serotype-2 vectors caused only a weak and highly transient innate immune response in the liver, suggesting that inflammatory reactions to AAV are negligible. Numerous animal studies have shown stable correction of genetic diseases by hepatic AAV gene transfer that may, in part, be because of the low innate immune profile of the vector, avoiding inflammatory signals. 4 In humans, hepatic gene transfer with AAV2 has been hampered by pre-existing adaptive immunity after natural infection in the form of neutralizing antibodies and capsid-specific CD8 ϩ T cells. 5 Numerous changes to capsid and vector genomes have been developed in recent years in attempts to improve gene transfer efficacy and possibly evade immunity. For example, AAV8 shows substantially higher transduction efficiency in mouse liver and reduced activation of capsid-specific T cells, and it facilitates tolerance induction to transgenes. 6,7 Furthermore, prevalence for neutralizing antibodies in humans is markedly lower to AAV8 than to AAV2. 8 In another set of investigations, replacing surfaceexposed tyrosine residues to phenylalanine has been shown to improve gene transfer for several serotypes. The resulting reduction in capsid phosphorylation in turn reduces accumulation in the cytoplasm (in favor of trafficking to the nucleus) and ubiquitination of capsid. 9 AAV2 gene transfer to hepatocytes was most improved by a combination of 3 Tyr-Phe changes in amino acid residues 444, 500, and 730. 10 Modifications of the recombinant AAV genome also can improve transduction rates. Being ss, the ssAAV genome has to be converted to a double-stranded form in the nucleus of an infected cell for transgene expression to occur. To overcome this ratelimiting step, self-complementary (sc)AAV vectors were developed by elimination of the terminal resolution site in one of the inverted terminal repeats (ITRs). 11 For such a genome to be packaged into capsid, the size of the expression cassette has to be further reduced to not exceed the packaging limit. Two groups reported optimized scAAV vectors for treatment of the X-linked bleeding disorder hemophilia B (coagulation factor IX deficiency) by liver gene transfer. 12,13 The hepatic microenvironment is more tolerogenic than that of many other tissues. 14 For example, we were able to tolerize hemophilia B mice to human factor IX (hF.IX) by hepatic ssAAV2 gene transfer. This protocol was successful in several strains, with the exception of C3H. 15 Nonetheless, AAV8 and AAV2(Y444/500/ 730F) vectors were able to...

Humoral and Cell-Mediated Immune Response, and Growth Factor Synthesis After Direct Intraarticular Injection of rAAV2-IGF-I and rAAV5-IGF-I in the Equine Middle Carpal Joint

“…Both type I and II interferons have been shown to increase in mice treated with AAV vectors; increases in IFN-a may be upregulated by AAV after activation of Toll-like receptor-9 (TLR9) in response to intracellular viral genomes, while increases in IFN-c appear to be associated with activated T cells. 37 In this study, there was no evidence of an IFN-a or IFN-c response in restimulated PBMCs, serum, or synovial fluid at any time point. In addition, no significant increases in IL-6 or IL-10 were seen after rAAV2 or rAAV5 administration.…”

CpG-depleted adeno-associated virus vectors evade immune detection

“…TLR9 has been implicated as a mediator of immunoreactivity toward single-stranded and self-complementary AAVs (scAAVs) due to its ability to recognize unmethylated CpG motifs present in the therapeutic expression cassettes packaged in an AAV capsid (15). Zhu et al demonstrated the role of TLR9/MyD88 signaling in mouse plasmacytoid dendritic cells as an important mediator of…”

“…Therefore, TLR9 has the potential to recognize unmethylated CpG motifs in the therapeutic expression cassettes packaged in an AAV capsid and induce innate and adaptive immunity (15). Previous reports revealed that innate immune recognition of the serotype AAV2 by plasmacytoid dendritic cells was mediated by TLR9 and dependent on MyD88 signaling, independent of the form of transgene (16).…”

“…Previous reports revealed that innate immune recognition of the serotype AAV2 by plasmacytoid dendritic cells was mediated by TLR9 and dependent on MyD88 signaling, independent of the form of transgene (16). This innate immune sensor has been implicated in promoting immune responses following self-complementary AAV gene transfer, but the underlying mechanisms have not been fully elucidated (15,17).…”

Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells

“…Their DNA genome and capsid structure is being sensed by Toll-like receptors 9 and 2, respectively, and humans often have preexisting immunity. [8][9][10][11][12][13] Activation of capsid-specific CD8…”