We assessed the efficiency of gene transfer by examining biopsy specimens of vector-injected biceps muscles and untreated biceps muscles on day 42 (in Patients 1, 3, 4, and 6) or day 90 (in Patients 2 and 5). In all the patients, vector DNA was detected in amounts ranging from 0.01 to 2.56 genome copies per diploid genome in the treated muscles but was not detected in the untreated contralateral biceps muscles. Myofibers expressing mini-dystrophin protein were not detected in the two biopsy specimens that were examined on day 90, but they were detected in two of the four specimens (those from Patients 3 and 6) that were examined on day 42. With the use of an antibody (MANDYS3) that detects the N-terminal of dystrophin, we found that Patient 3 had three or four dystrophin-positive fibers, and Patient 6 had one positive fiber (data not shown). These fibers were negative for dystrophin on immunohistochemical staining with an antibody that detects the C-terminal, which is removed when dystrophin complementary DNA is miniaturized to accommodate the small insertion capacity of the AAV.
Failure to establish long-term transgene expression prompted us to measure cell-mediated immune responses to mini-dystrophin in all six patients. Mini-dystrophin–specific T cells were not detected at any time in Patients 1 and 3 (who received low doses of vector), as assessed with the interferon-γ ELISPOT assay (). Patient 2, who also received a low dose of vector, had robust T-cell activity against peptide pool MDP2 on the 15th day after vector treatment (). The response peaked on day 30, slowly declined during the next 3 months, and was intermittently positive after day 120 (). T-cell responses were substantially delayed in Patients 5 and 6 (, respectively), who received high doses of vector, and exceeded the detection threshold of 50 spot-forming cells per 1 million PBMCs only on day 60. A T-cell response to MDP3 was unexpectedly detected before vector treatment in Patient 4 and was intermittently positive throughout 2 years of follow-up (). Delivery of a high dose of the gene-therapy vector to the biceps muscle of Patient 4 did not elicit the rapid increase in dystrophin-specific T-cell activity that was observed in Patient 2.
Cellular immunity was further characterized in Patient 2 and Patient 5 to better understand differences in the timing and duration of the response to mini-dystrophin. The delayed, transient T-cell response detected in Patient 5 on day 60 targeted three discrete epitopes. One exon-7 epitope was recognized by HLA-B*1801–restricted CD8+ T cells (). CD4+ T cells recognized two epitopes that localized to exon 8 () and exon 6 (). Because exons 3 through 17 were deleted from the dystrophin gene in this patient, we concluded that CD4+ and CD8+ T cells were primed by therapeutic mini-dystrophin that incorporated missing exons 3 through 12. Striking amino acid homology was observed between the exon-6 and exon-7 epitopes of dystrophin and corresponding sequences of β-spectrin and utrophin, cellular proteins that are also involved in muscle-force contraction and expressed at normal levels in dystrophic muscle. For instance, the exon-6 epitope presented by HLA-DQA1*0505 and HLA-DQB1*0301 () was mapped to a segment of dystrophin composed of 10 amino acids (, with amino acids denoted by single-letter symbols) — 163-WSDGLALNAL-172 — that differed from β-spectrin at only two positions (). T cells from Patient 5 recognized the dystrophin epitope but not the β-spectrin homologue containing the S164R and L169F substitutions, which suggests that homology between these highly related cellular proteins restricted, but did not eliminate, the pool of nonself epitopes available for immune recognition.
Characterization of Dystrophin-Specific Cellular Immune Responses in Patient 5
The rapid MDP2-specific response observed in Patient 2 was mediated by CD4+ T cells, since depletion of this subgroup from PBMCs abrogated interferon-γ
production (). The epitope was localized to amino acids 2809 to 2829 of exon 57 (). Exon 57 should be frame-shifted because of the exon-50 deletion carried by this patient. Alternative splicing or a second mutation in the DMD
gene can restore in-frame expression of functional dystrophin in rare revertant muscle fibers.7
Revertant fibers were visualized in the muscle specimen from Patient 2 with the use of antibodies directed against dystrophin exons 55–56 and 59 (). Exon 57 encoding the T-cell epitope was therefore expressed in the correct reading frame, despite the exon-50 deletion. We postulated that T cells spontaneously primed by revertant fibers were present before treatment with vector and accelerated the pace of the cellular immune response to therapeutic mini-dystrophin. Indeed, a robust interferon-γ
response was detected when cryopreserved PBMCs collected before and after vector treatment were stimulated with the peptide epitope spanning amino acids 2809 through 2829 of exon 57 (). Dystrophin-specific antibodies were not detected in the serum of any study patients, including Patient 2 (data not shown).
Mini-Dystrophin Cellular Immune Responses in Patient 2
The preexisting cellular immunity to dystrophin in Patient 4, who had a deletion at exons 49 through 54 (), was also associated with the expression of revertant muscle fibers. The response mediated by CD8+ T cells (Fig. 1 in the Supplementary Appendix
) was directed against MDP3 that contains peptides from the R24, H4, and cysteine repeat regions encoded by exons 59 through 70 (). Revertant fibers expressing exons 59 through 70 were detected in biceps muscle with the use of exon-specific antibodies; in-frame dystrophin sequences containing this epitope were expressed in the skeletal muscle of Patient 4 (, and Fig. 2 in the Supplementary Appendix
). Expression of HLA class I proteins was greater in the treated biceps muscle than in the untreated muscle in Patient 4, indicating the potential for CD8+ T-cell recognition of myocytes (Fig. 3 in the Supplementary Appendix
). A similar increase in HLA class I expression by myocytes was observed in Patient 5, in whom CD8+ T cells were directed against nonself epitopes encoded by the transgene (Fig. 3 in the Supplementary Appendix