AbstractIn 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.

Myelomeningocele (MMC) is a common and devastating malformation. Although fetal surgical closure may improve outcome, a less invasive approach that can be applied earlier in gestation is desirable. The objective of this study was to evaluate the therapeutic feasibility of a tissue engineering approach for prenatal coverage of MMC. A gelatin hydrogel composite combining a gelatin sheet and gelatin sponge was prepared with or without basic fibroblast growth factor incorporation, and applied prenatally to retinoic-acid-induced fetal MMC in the rat model. Most of the composites were adherent to the MMC within the amniotic fluid environment with the help of cyanoacrylate adhesive. Histological examination revealed cells layered over the composites with associated extracellular matrix as well as cellular ingrowth into the sponges. The layer over the composite was composed of mixed nonepithelial and epithelial cells with the extracellular matrix consisting of collagen type I and hyaluronic acid. The tissue inside the sponge consisted of nonepithelial cells and hyaluronic acid. Epidermal ingrowth underneath the sponges and neovascularization into the sponges occurred and were significantly increased by the incorporation of basic fibroblast growth factor. Although further development is needed, this study supports the therapeutic potential of a tissue engineering approach for prenatal coverage of MMC.

Myelomeningocele (MMC) is a common and devastating malformation. As an alternative to fetal surgical repair, tissue engineering has the potential to provide a less invasive approach for tissue coverage applicable at an earlier stage of gestation. We have previously evaluated the use of gelatin hydrogel composites composed of gelatin sponges and sheets as a platform for tissue coverage of the MMC defect in the retinoic acid induced fetal rat model of MMC. In the current study, we compare our previous composite with gelatin microspheres as a scaffold for tissue ingrowth and cellular adhesion within the amniotic fluid environment. We also examine the relative efficacy of various bioactive protein coatings on the adhesion of amniotic fluid cells to the construct within the amniotic cavity. We conclude from this study that gelatin microspheres are as effective as gelatin sponges as a scaffold for cellular ingrowth and amniotic fluid cell adhesion and that collagen type I and fibronectin coatings enhance amniotic fluid cell adhesion to the gelatin-based scaffolds. These findings support the potential for the development of a tissue-engineered injectable scaffold that could be applied by ultrasound-guided injection, much earlier and less invasively than sponge or sheet-based composites.

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