Results from several experimental systems suggest that cells from one tissue type can form other tissue types after transplantation. This could be due to the presence of multipotential or several types of adult stem cells in donor tissues, or alternatively, to fusion of donor and recipient cells. In a model of tyrosinaemia type I, mice with mutations in the fumarylacetoacetate hydrolase gene (Fah-/-) regain normal liver function after transplantation of Fah+/+ bone marrow cells, and form regenerating liver nodules with normal histology that express Fah. Here we show that these hepatic nodules contain more mutant than wild-type Fah alleles, and that their hepatocytes express both donor and host genes, consistent with polyploid genome formation by fusion of host and donor cells. Using bone marrow cells marked with integrated foamy virus vectors that express green fluorescent protein, we identify common proviral junctions in hepatic nodules and haematopoietic cells. We also show that the haematopoietic donor genome adopts a more hepatocyte-specific expression profile after cell fusion, as the wild-type Fah gene was activated and the pan-haematopoietic CD45 marker was no longer expressed.

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A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Here we show the first evidence of widespread transduction of both cardiac and skeletal muscles in an adult mammal, after a single intravenous administration of recombinant adenoassociated virus pseudotype 6 vectors. The inclusion of vascular endothelium growth factor/vascular permeability factor, to achieve acute permeabilization of the peripheral microvasculature, enhanced tissue transduction at lower vector doses. This technique enabled widespread muscle-specific expression of a functional micro-dystrophin in the skeletal muscles of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy. We propose that these methods may be applicable for systemic delivery of a wide variety of genes to the striated muscles of adult mammals.Human mortality and quality of life are significantly affected by diseases of the striated musculature. Genetic treatments that are being developed for conditions such as heart disease, aging-associated muscle wasting and the muscular dystrophies have been limited by an inability to achieve widespread and efficient gene transfer to the heart and dispersed skeletal muscles of an adult organism 1-4 . For example, anesthesia, invasive surgery and hazardous cofactors are required to transduce varying fractions of the cardiomyocyte population efficiently 1,5 . Similarly, the transfer of genes to the muscles of individual limbs using various vectors requires either direct injection of individual muscles, or complex surgical procedures performed under anesthesia to distribute vectors via the circulation 2-4,6-10 . Here we describe a simple and highly efficient method to transfer genes systemically to the cardiac and skeletal muscles of adult mammals. This approach uses intravenous administration of recombinant adeno-associated virus pseudotype 6 (rAAV6-pseudotyped vectors) 11 , which are extremely effective at transducing skeletal muscles after intramuscular injection 12 .Correspondence should be addressed to J.S.C. (jsc5@u.washington.edu).. 4 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Medicine website. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Medicine website for details). NIH Public Access RESULTS Systemic transduction of skeletal muscles by rAAV6First, we examined the potential for systemic gene transfer after intravenous administration of rAAV6 vectors at the whole-body level in young adult (6-8 wk) C57Bl/10J mice (Fig. 1). The muscles of mice examined 11 days after administration through the tail vein of ~2 × 10 11 vector genomes of rAAV6 vector containing a CMV-lacZ expression cassette, did not show obvious exogenous β-galactosidase (...

Adeno-associated viruses (AAV) are promising gene therapy vectors that have little or no acute toxicity. We show that normal mice and mice with mucopolysaccharidosis VII (MPS VII) develop hepatocellular carcinoma (HCC) after neonatal injection of an AAV vector expressing b-glucuronidase. AAV proviruses were isolated from four tumors and were all located within a 6-kilobase region of chromosome 12. This locus encodes several imprinted transcripts, small nucleolar RNAs (snoRNAs), and microRNAs. Transcripts from adjacent genes encoding snoRNAs and microRNAs were overexpressed in tumors. Our findings implicate this locus in the development of HCC and raise concerns over the clinical use of AAV vectors.

Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this ‘missing self’ response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8+ T cells, do not bind anti-HLA antibodies, and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.

BackgroundInherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells.Methodology/Physical FindingsIn this report we have used a similar method to direct induced pluripotent stem cells (iPS) from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS) after labeling photoreceptors with a lentivirus driving GFP from the IRBP cis-regulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers.ConclusionsThis report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells.

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Stable transduction of mammalian cells typically involves random integration of viral vectors by non-homologous recombination. Here we report that vectors based on adeno-associated virus (AAV) can efficiently modify homologous human chromosomal target sequences. Both integrated neomycin phosphotransferase genes and the hypoxanthine phosphoribosyltransferase gene were targeted by AAV vectors. Site-specific genetic modifications could be introduced into approximately 1% of cells, with the highest targeting rates occurring in normal human fibroblasts. These results suggest that AAV vectors could be used to introduce specific genetic changes into the genomic DNA of a wide variety of mammalian cells, including therapeutic gene targeting applications.