Investigating Immune Responses to the scAAV9- HEXM Gene Therapy Treatment in Tay-Sachs Disease and Sandhoff Disease Mouse Models

Abstract

GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme β-hexosaminidase A (HexA). HexA consists of an α- and β-subunit; a deficiency in either subunit results in Tay-Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and "non-self"-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed "non-self" proteins, and potentially improve treatment efficacy.

Keywords: GM2; HexM; Sandhoff; Tay-Sachs; capsid; gangliosidosis; immunocompetence; murine; scAAV9-HEXM; transgene.

Figure 1

Hex peptide T cell responses in TSD mice. (A) ELISpot IFN-γ secretion in response to the total HexM 52 peptide pool (See Supplementary Materials) in Het and KO TSD mice 3 weeks and 6 weeks post injection (PI). HEXM-treated mice showed significantly higher number of spots to HexM protein compared to vehicle-treated mice (ANOVA main effect of treatment, p < 0.0001). (B) Comparison of IFN-γ secretion spots in response to novel HexM peptides (i.e., not homologous with either the mouse or human HexA) in male Het and KO TSD mice 3 weeks and 6 weeks. HEXM-treated mice showed significantly higher number of spots to HexM protein compared to vehicle-treated mice (ANOVA main effect of treatment, p < 0.001). (C) Number of spots in response to exposure with peptides with 100% homology between humans and mice in male Het and KO TSD mice 3 weeks and 6 weeks PI. HEXM-treated mice showed significantly higher number of spots to HexM protein compared to vehicle-treated mice (ANOVA main effect of treatment, p < 0.01), and there was a tendency for KO TSD mice to show a greater response to these peptides at 6 weeks than at 3 weeks (p < 0.05, post hoc tests). (D) Number of spots in response to the HexM peptide pool specific to human HexA in male Het and KO TSD mice 3 weeks and 6 weeks PI. HEXM-treated mice showed significantly higher number of spots to HexM protein compared to vehicle-treated mice (ANOVA main effect of treatment, p < 0.001). In summary, both heterozygous and KO TSD mice treated with scAAV9-HexM had a T-cell response to all types of peptides in the hex peptide pool. M = male, F = female, wks = weeks, PI = post injection, N per group = 3, error bars = SEM, **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p < 0.05.

Figure 2

HexM-specific T cell response in SD mice. ELISPot IFN-γ secretion in response to HexM stimulated splenocytes of SD mice 3 weeks and 9 weeks post injection. Mice receiving scAAV9-HexM and euthanized 3 weeks post injection showed significantly higher number of spots in response to HexM peptides, compared to SD mice receiving vehicle or HexM protein injections only, with or without adjuvant (p < 0.0001); differences among the latter groups of SD mice were not significant. †Mice receiving one or two injections of scAAV9-HexM and euthanized 9 weeks post the first (or only) injection, had fewer spots than mice receiving scAAV9-HexM euthanized 3 weeks post injection (p < 0.01 post hoc), but a higher number of spots than mice receiving vehicle or vehicle with adjuvant (p = 0.06 post hoc using Tukey–Kramer corrections for p values.) KO = knock out SD mice, Adj = adjuvant, Euth = euthanized, wks = weeks, PI = post injection, error bars = SEM, n per group as shown, ns = not significant, **** = p < 0001.

Figure 3

ELISpot IFN-γ secretion in response to AAV9 capsid peptides in male and female Het and KO TSD mice 3 weeks and 6 weeks post vehicle or scAAV9-HEXM injection (PI). Mice receiving scAAV9-HEXM showed significantly higher number of spots to AAV9 capsid peptides compared to vehicle-injected mice (ANOVA main effect of treatment, p < 0.0001). Differences between males versus females and Het versus KO mice were not statistically significant. A post hoc analysis found that among scAAV9-HEXM-treated mice, the T cell response in splenocytes harvested from mice at 6 weeks was less than that in splenocytes harvested from mice at 3 weeks (blue versus red bars, right half of graph, p < 0.01). M = male, F = female, wks = weeks, PI = post injection. Error bars = SEM, N per group = 3, **** = p < 0001.

Figure 4

Anti-HexM antibody levels in the sera of TSD mice. HexM-specific B cell response in TSD KO and Het mice at 3 weeks and 6 weeks post scAAV9-HEXM injections. TSD mice given vehicle injections did not develop anti-HexM antibodies in the sera. Mice treated with scAAV9-HEXM developed significantly higher levels of anti-HexM antibodies, compared to those receiving vehicle. Among mice receiving scAAV9-HexM, those receiving a second injection with HexM protein after 3 weeks, then terminated at 6 weeks post- vector injection, had antibody levels more than double those that were terminated at 3 weeks post-vector injection (p < 0.001). There were no significant differences between Het and KO mice, nor between males and females (not shown). wks = weeks, PI = post-vector injection, **** = p < 0.001, n per group = 6 (3 males and 3 females), error bars = SEM.

Figure 5

Anti-HexM antibody levels in sera of SD mice. HexM-specific B cell response in SD KO and Het mice at the endpoint (3 weeks post injection 1 or 6 weeks post injection 2). High levels of anti-HexM antibodies were observed in both Het and KO mice given HexM purified protein with adjuvant. Vehicle-injected mice showed low levels of anti-HexM antibodies. Euth = euthanized, wks = weeks, PI = post injection, error bars = SEM, **** = p < 0001.

Figure 6

Vector biodistribution of scAAV9-HEXM in the brain of TSD mice. Biodistribution of the scAAV9-HEXM viral vector, in the brain, 3 weeks and 6 weeks post scAAV9-HEXM injection in TSD mice. (a) Comparison of scAAV9-HEXM treated to vehicle injected mice. (b) comparison of Het and KO mice. A lower biodistribution was obtained in brain samples from KO mice terminated at the later time point compared to the earlier time point, with this observation near to traditional significance levels (p = 0.055). M = male, F = female, wks = weeks, PI = post injection.

Figure 7

Vector biodistribution of scAAV9-HEXM in the liver of TSD mice. Biodistribution of the scAAV9-HEXM viral vector, in the liver, 3 weeks and 6 weeks post scAAV9-HEXM injection in TSD mice. (a) Comparison of scAAV9-HEXM treated to vehicle injected mice. (b) comparison of Het and KO mice. A lower biodistribution was evident in liver samples from mice terminated at the later time point compared to the earlier time point, significantly so in the KO mice (* = p < 0.05). M = male, F = female, wks = weeks, PI = post injection.

Figure 8

Study timeline in weeks of (a) TSD study and (b) SD study. (a) TSD mice were given the first injection at 8 weeks of age, and the second injection at 11 weeks of age. Mice were euthanized either at 11 weeks (n = 24) or at 14 weeks (n = 24). (b) SD mice were given their first injection at 6 weeks of age and their second injection at 9 weeks of age. Mice were euthanized at 9 weeks (n = 3) or at 15 weeks (n = 34).