Gene therapy offers a promising therapy for patients affected with an array of genetic diseases. Adeno-associated virus (AAV) vector efficiently transduces dividing or nondividing cells through both local and systemic administration, providing multiple strategies to treat genetic diseases. While thought to be minimally immunogenic—an attribute that allows for sustained transgene expression from AAV vectors in some experimental models—adaptive immune responses have been observed and, in some cases, implicated in the loss of transgene expression.

FIGURE 1: CpG+ and CpG- LacZ AAV expression plasmids express at quantitatively comparable levels.

Potential immune toxicity of AAV is complex and multifactorial. Activation of de novo and preexisting cellular and humoral immunoreactivity toward viral capsid and encoded transgene antigens has the potential to limit long-term transgene expression. Toll like receptors (TLRs) recognize an array of pathogen-associated molecular patterns (PAMPs) of infectious microbes and are critically important for the induction of proinflammatory immune responses that initiate innate and adaptive immune responses. TLRs are highly expressed on innate immune cells such as macrophages and mast cells, dendritic cells, and to a lesser extent, B and T cells, endothelial cells, epithelial cells, and fibroblasts. TLR9 senses the major pathogenic DNA viruses including herpes simplex virus (HSV), EBV, CMV, adenovirus, and human papilloma virus. TLR9 is found almost exclusively within intracellular compartments such as endosomes. This receptor is capable of recognizing unmethylated bacterial and viral CpG motifs initiating signaling pathways that activate the transcription factors NF-kB, interferon (IFN) regulatory factors (IRFs), and activating protein-1. TLR9 signaling can lead to the production of inflammatory cytokines IL-6, TNF-alpha, type I IFNs, and IL-1-beta, initiating both innate and adaptive immune responses. In addition, CpG motifs have been demonstrated to induce chemokines, major histocompatibility complex type II (MHC II) molecules, and costimulatory ligands on monocytes, which have been postulated to act as nonprofessional antigen presenting cells (APCs).

In vivo studies in wild type (WT) mice by the inventors of NxGEN technology demonstrate that intramuscular gene transfer with the AAV vector, AAVrh32.33, induces a robust adaptive immune response toward both capsid and transgene antigen, heavy cellular infiltrate, and a loss of detectable transgene expression at days 35 and 60 after administration. This robust response is dependent on TLR9 as mice genetically engineered to lack the TLR9 receptor (TLR9KO mice) injected intramuscularly with AAVrh32.33 exhibit prolonged transgene expression and fail to show an inflammatory response toward transgene and capsid antigen.

To determine whether CpG-depleted AAVrh32.33 vectors would abrogate the robust cellular immune response and transgene loss observed in the WT mice, the inventors compared the performance of 2 vectors that differed only by the abundance of CpG motifs. Both vectors contained a cytoplasmic lacZ open reading frame expressed from a mammalian-derived promoter flanked by AAV2 inverted terminal repeats (ITRs).

FIGURE 2: AAVCpG+ transduced muscle exhibits a progressive loss of detectable β-gal expression, while the muscle sections from CpG-depleted AAV-transduced mice display robust and stable transgene expression.

The CpG+ vector contained 16 CpGs in the ITRs and 308 CpGs in the lacZ gene for a total of 324 CpGs. The CpG vector was void of CpGs in the lacZ gene, meaning its total CpG content was 16. CpG-depleted plasmid packaging was up to 80% as efficient as WT CpG-containing plasmids. To assay expression levels, HeLa cells were transfected with CpG+ and CpG– AAV expression plasmids. Both plasmids express at a quantitatively comparable level (Figure 1).

FIGURE 3: A significant reduction in the percentage of LacZ-responsive CD8+ T cells was observed in mice that received the AAVCpG- but not the AAVCpG+ vector.

To test the hypothesis that CpG-depleted AAVrh32.33 vectors would exhibit prolonged transgene expression, gastrocnemius tissues from WT mice injected intramuscularly with 1 x 1011 genome copies (GC) of AAVrh32.33CpG+ (AAVCpG+) or AAVrh32.33CpG– (AAVCpG–) expressing a cytoplasmic β-gal protein were stained with X-gal (Figure 2A-D). AAVCpG+ transduced muscle exhibited a progressive loss of detectable β-gal expression, while the muscle sections from CpG-depleted AAVrh32.33LacZ-transduced mice displayed robust and stable transgene expression. Hence, the steady loss of LacZ transgene expression following AAVrh32.33LacZ gene transfer is dependent on viral genome CpG motifs consistent with the role of toll-like receptor 9 (TLR9) activation of innate immunity.

Transgene stability observed in the AAVCpG-transduced muscle sections demonstrates an abrogated adaptive immune response toward transgene and capsid antigen. To assess the requirement for CpG motifs in the induction of an adaptive immune response toward AAVrh32.33 in the modified vectors, MHC I tetramer stain and ELISPOT assays were used to quantify transgene-reactive FIGURE 4: A significant decrease of primed transgene and capsid antigen-reactive IFN-γ ELISPOT responses was observed in mice that received the AAVCpG- but not AAVCpG+ vector.

CD8+ T cells and primed transgene and capsid responsive interferon gamma (IFN-γ) producing T cells (Figures 3 and 4). Mice that received the CpG depleted AAVrh32.33LacZ vector exhibited a significant reduction (P ≤ 0.05) in the percentage of LacZ-responsive CD8+ T cells compared with mice that received the CpG+ vector (Figure 3). Further, a significant decrease of primed transgene and capsid antigen–reactive IFN-γ ELISPOT responses was observed in mice that received the AAVCpG– but not AAVCpG+ vector (Figure 4). These findings show the remarkable ability of AAV gene therapy vectors built using NxGEN technology to escape the immune responses that have challenged the field of gene therapy for decades, leading to safe and effective gene transfer.

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