Emerging Immunogenicity and Genotoxicity Considerations of Adeno-Associated Virus Vector Gene Therapy for Hemophilia

Abstract

Adeno-associated viral (AAV) vector gene therapy has shown promise as a possible cure for hemophilia. However, immune responses directed against AAV vectors remain a hurdle to the broader use of this gene transfer platform. Both innate and adaptive immune responses can affect the safety and efficacy of AAV vector-mediated gene transfer in humans. These immune responses may be triggered by the viral capsid, the vector's nucleic acid payload, or other vector contaminants or excipients, or by the transgene product encoded by the vector itself. Various preclinical and clinical strategies have been explored to overcome the issues of AAV vector immunogenicity and transgene-related immune responses. Although results of these strategies are encouraging, more efficient approaches are needed to deliver safe, predictable, and durable outcomes for people with hemophilia. In addition to durability, long-term follow-up of gene therapy trial participants will allow us to address potential safety concerns related to vector integration. Herein, we describe the challenges with current methodologies to deliver optimal outcomes for people with hemophilia who choose to undergo AAV vector gene therapy and the potential opportunities to improve on the results.

Keywords: adeno-associated virus vector; cellular immunity; gene therapy; genome integration; hemophilia; humoral immunity; immunogenicity; immunologic tolerance; innate immunity; liver transduction; neutralizing antibody.

Figure 1

Immune response to AAV gene transfer. In the first gene transfer clinical trial hepatic gene transfer for hemophilia B, an AAV2 vector expressing the human FIX transgene was administered via the hepatic artery. Results in mice and other preclinical animal models showed persistence of transgene expressing and no immune responses following AAV gene transfer. In humans, transgene expression was initially detected but started to decline after 4 to 6 weeks concomitant to an increase in liver enzymes and the detection of T-cell reactivity against the vector capsid. Subsequent studies [16] showed that cytotoxic T-lymphocyte (CTL) expansion detected in the peripheral blood was triggered by the administration of the AAV2 vector in humans and was likely responsible for the clearance of AAV-transduced hepatocytes. As noted, CD8+ T-lymphocyte expansion was not observed in preclinical animal models, in which stable transgene expression had been observed [16]. AAV, adeno-associated virus; FIX, factor IX.

Figure 2

Mechanisms of potential immune responses to AAV vectors [30]. (A) Hepatocytes transduced with AAV vectors (a) express the therapeutic protein but also present capsid-derived peptides (yellow triangles) via their MHC class 1 molecules (b). A fraction of the vector dose enters proximal lymph nodes and is taken up by pDCs (c), where vector DNA is processed in the lysosome and promotes the production of proinflammatory cytokines, and by cDCs (d), where vector capsid-derived peptides (red circles) are presented by MHC class 2 molecules, recruiting capsid-specific CD4+ T-cell help. These events lead to licensing and maturation of cDCs and activation of capsid-specific CD8+ CTLs (e) that proliferate, migrate to the liver, and eliminate transduced hepatocytes (f). (B) An ideally designed AAV vector with low immunogenicity (g) would similarly transduce hepatocytes but would not activate innate immunity. CTLs would not be formed, transduced hepatocytes would not be eliminated, and cell-surface capsid peptide presentation would wane (h). In both scenarios, AAV vectors activate the humoral arm of the immune response (i), leading to capsid antibodies. Adapted from wright [30]. AAV, adeno-associated virus; ALT, alanine aminotransferase; AST, aspartate aminotransferase; cDC, conventional dendritic cell; CTL, cytotoxic T lymphocyte; IFN, interferon; IL, interleukin; MHC, major histocompatibility complex; PAMP, pathogen-associated molecular pattern; pDC, plasmatoid dendritic cell; TLR9, Toll-like receptor 9; rAAV, recombinant adeno-associated virus; tx, therapeutic.

Figure 3

Potential limitations of gene transfer with AAV vectors [47]. Several potential immunologic-related issues to the AAV vector platform are emerging (white box): (1) Vector immunogenicity: the presence of neutralizing antibodies (NAbs) against the AAV capsid can prevent or limit cell transduction, whereas cytotoxic CD8+ T-cell responses can eliminate AAV-transduced cells that present AAV capsid antigens loaded on MHC-I complexes. (2) Potency and efficacy: the efficiency with which AAV vectors infect and transduce into the desired target cells can impact therapeutic doses and efficacy. (3) Genotoxicity: although rare, the integration of the AAV vector DNA into the genome of the infected cell may have genotoxic effects. (4) Persistence: the episomal AAV genome in the nucleus of the infected cells can be lost in conditions of cell proliferation (such as liver growth), which may impact therapeutic efficacy. AAV, adeno-associated virus; ER, endoplasmic reticulum; MHC-I, major histocompatibility complex class I molecule; sc, self-complementary; ss, single strand; TLR, Toll-like receptor.

Figure 4

Assays to test for anti-AAV antibodies and neutralizing factors. (A) The cell-based transduction inhibition assay measures the ability of plasma samples to reduce the transduction of a cell line by a recombinant adeno-associated virus (rAAV) vector carrying a reporter transgene such as luciferase. In the presence of antibodies, the luciferase-reported fluorescence may be reduced. (B) Total anti-AAV antibodies assays on human plasma using a bridging electrochemiluminescence assay or a classic capture assay. AAV capsids are coated to a well, plasma samples are added after blocking, and AAV-specific antibodies are detected using ruthenylated AAV capsids (for electrochemiluminescence assay) or an anti-IgG antibody is added (for the capture assay). max, maximum; min, minimum.

Figure 5

Mechanism of action of IdeS. IdeS is an IgG-degrading enzyme derived from Streptococcus pyogenes proposed as a strategy to overcome the limitation of neutralizing antibodies (NAbs) to AAV. IdeS is an endopeptidase that cleaves human IgG into F(ab′)2 and Fc fragments, thus reducing the neutralization activity of anti-AAV antibodies. AAV, adeno-associated virus; IdeS, imlifidase; IgG, immunoglobulin G; Fc, fragment crystallizable region; F(ab′)2, 2 antigen-binding (Fab) regions.

Figure 6

Antigen-specific transgene tolerance. Hepatic gene transfer with adeno-associated virus (AAV) vectors induces tolerance by multiple mechanisms, which include programmed cell death of CD4⁺ T-helper cells and the induction of FoxP3⁺ T_reg. The initial presentation of antigens to the liver draining portal/celiac lymph nodes and the liver resident antigen-presenting cells (APCs) plays an important role in the induction of liver tolerance. DC, dendritic cell; Fas, Fas cell surface death receptor; FasL, Fas ligand; IL, interleukin; MF, macrophage; TCR, T-cell receptor; Treg, T regulatory cell.