Neutralizing Antibodies Against Adeno-Associated Viral Capsids in Patients with mut Methylmalonic Acidemia

Abstract

Isolated methylmalonic acidemia (MMA), a group of autosomal recessive inborn errors of metabolism, is most commonly caused by complete (mut(0)) or partial (mut(-)) deficiency of the enzyme methylmalonyl-CoA mutase (MUT). The severe metabolic instability and increased mortality experienced by many affected individuals, especially those with mut(0) MMA, has led centers to use elective liver transplantation as a treatment for these patients. We have previously demonstrated the efficacy of systemic adeno-associated viral (AAV) gene delivery as a treatment for MMA in a murine model and therefore sought to survey AAV antibody titers against serotypes 2, 8, and 9 in a group of well-characterized MMA patients, accrued via a dedicated natural history study ( clinicaltrials.gov ID: NCT00078078). Plasma samples provided by 42 patients (8 mut(-) and 34 mut(0); 10 had received organ transplantation), who ranged in age between 2 and 31 years, were analyzed to examine AAV2 (n = 35), AAV8 (n = 41), and AAV9 (n = 42) antibody titers. In total, the seroprevalence of antibodies against AAV2, AAV8, or AAV9 was 20%, 22%, and 24%, respectively. We observed a lower-than-expected seropositivity rate (titers ≥1:20) in the pediatric MMA patients (2-18 years) for both AAV2 (p < 0.05) and AAV8 (p < 0.01) neutralizing antibodies (NAbs) compared with historical controls. Those with positive NAb titers were typically older than 18 years (p < 0.05 all serotypes) or had received solid organ transplantation (p < 0.01 AAV8, AAV9). The mut(0) patients who had not been transplanted (n = 24)-that is, the subset with the greatest need for improved treatments-represented the seronegative majority, with 21 out of 24 patients lacking Abs against all AAV capsids tested. The unexpected lack of NAbs against AAV in this patient population has encouraging implications for systemic gene delivery as a treatment for mut MMA.

Figure 1.

(A) AAV antibody seroprevalance within the entire MMA cohort (n = 42) that includes both nontransplanted and transplanted (liver, kidney, and/or combined liver–kidney; n = 10) patients. The overall seroprevalance, defined as ≥1:5, is depicted (20–24%) along with the titer distribution of antibodies against AAV2 (n = 35), AAV8 (n = 41), and AAV9 (n = 42) capsids. The seroprevalence is subdivided into titer dilution levels from weak (1:5) NAb titers to strong (1:320). (B) AAV antibody seropositivity in the subgroup of patients with mut⁰ MMA who were not transplanted (n = 24), which shows low NAb prevalence (4–10%) for the cohort of severe, nontransplanted patients who would be ideal candidates for gene therapy. AAV, adeno-associated viral; Ab, antibody; MMA, methylmalonic acidemia; NAb, neutralizing antibody.

Figure 2.

(A) Seropositivity, as defined as ≥1:20, in the pediatric MMA cohort compared with historical controls. There was a lower-than-expected AAV2 (p = 0.0066) and AAV8 (p = 0.0408) NAb seroprevalance in MMA individuals ≤18 years. (B) Neutralizing antibody prevalence to AAV serotypes 2, 8, and 9 by age. The seroprevalance of AAV2, AAV8, and AAV9 NAbs between children and adults was significantly different for all serotypes with a higher incidence of seropositivity in adults (7/11). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Subgrouping of the MMA cohort by enzymatic subtype (A) and transplant status (B). (A) The seroprevalance was similar in mut⁻ and mut⁰ groups (AAV2 p = 0.1952; AAV8 p = 1.0; AAV9 p = 1.0). (B) The seroprevalence was significantly different in the transplanted and nontransplanted groups for AAV8 (p = 0.0008) and AAV9 (p = 0.0016) but not AAV2 (p = 0.3007) with 6/10 of the transplanted patients seropositive against AAV2, 8, and/or 9. **p < 0.01, ***p < 0.001.