Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer

Abstract

In previous work we transferred a human factor IX-encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer.

Figure 1

Evidence of F.IX protein in subject F muscle. (A) Western blot for human F.IX on protein extracted from tissue removed at bedside on patient expiry (top half) and at autopsy (bottom half); A indicates autopsy; R, right leg; L, left leg; F.IX, Benefix recombinant hF.IX control. (B) H&E stain of patient-injected muscle showing normal histology. (C-D) F.IX IHC of strongly F.IX-positive (C, same sample boxed in red from panel A) and very weakly positive (D, same sample boxed in black from panel A) tissue sections of injected patient muscle. (C-F) Magnification: ×20; red stars indicate F.IX-expressing muscle fibers; and white stars, no F.IX production. (E-F) F.IX IF of strongly F.IX-positive (E, same sample boxed in red from panel A) and very weakly positive (F, same sample boxed in black from panel A) sections of injected patient muscle.

Figure 2

Evidence of F.IX RNA, AAV vector DNA in subject F muscle. (A) Evidence of active transcription of F.IX RNA. Two independent primer pair PCR amplifications (216-bp and 408-bp amplicons) specific for human F.IX cDNA on patient muscle cDNA derived from isolated RNA. Three-week post-AAV2-CMV-F.IX–injected mouse (C57BL/6) muscle RNA-derived cDNA serves as a positive control. (B) Evidence of AAV vector genome in patient muscle. Primers amplifying a 242-bp genomic DNA segment from the CMV promoter of the vector to the exon 1/intron 1 junction of the transgene cassette reveal persistence of vector in injected patient muscle DNA. Three-week post-AAV2-CMV-F.IX–injected quadriceps mouse muscle genomic DNA serves as a positive control, while the contralateral uninjected quadriceps muscle serves as a negative mouse control.