Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes.

Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction.

Tropism-modified AAV vectors overcome barriers to successful cutaneous therapy.

Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvß8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy.

Gene transfer into human cord blood-derived CD34(+) cells by adeno-associated viral vectors.

OBJECTIVE: Bone marrow-derived CD34(+) cells are currently used in clinical trials in patients with ischemic heart disease. An option to enhance activity of injected progenitors may be offered by genetic engineering of progenitor cells with angiogenic growth factors. Recombinant adeno-associated viral vectors (rAAV) have emerged as a leading gene transfer systems. In contrast to other vector systems in use for genetic engineering of CD34(+) cells, rAAV-mediated gene expression does not depend on vector integration. This is relevant for application in regenerative medicine of ischemic tissues, where transient transgene expression is likely sufficient to achieve therapeutic benefits. MATERIALS AND METHODS: We compared three different human AAV serotypes, packaged as pseudotypes by a helper virus-free production method, for their transduction efficiency in human cord blood-derived CD34(+) cells. We further assessed the impact of vector genome conformation, of alpha(v)beta(5) and alpha(5)beta(1) integrin availability and of the transcription-modulating drugs retinoic acid and Trichostatin A on rAAV-mediated human CD34(+) cell transduction. RESULTS: We provide, for the first time, evidence that hCD34(+) cells can be reproducibly transduced with high efficiency by self-complementary rAAV2 without inducing cytotoxicity or interfering with their differentiation potential. We further show the involvement of alpha(5)beta(1) integrin as a crucial AAV2 internalization receptor and a function for transcription-modulating drugs in enhancing rAAV-mediated transgene expression. CONCLUSION: This study represents a first step toward translation of a combined cellular/rAAV-based therapy of ischemic disease.

P2X(3) receptor expression at early stage of mouse embryogenesis.

In this study we examined the expression of P2X(3) receptor in mouse embryos from E9.5 to E14.5 using immunohistochemistry. We found a uniform labeling in the developing trigeminal and dorsal root ganglia (DRG), while adult DRG and trigeminal ganglia expressed P2X(3) only in small-diameter neurons. In the brainstem, the mesencephalic trigeminal and facial motor nuclei were immunoreactive for P2X(3). P2X(3) was also transiently expressed in the developing brain, and precursors of spinal motor neurons. We also detected immunolabeling in the paravertebral sympathetic chain ganglia, in the sympathoadrenal cells and in non-neural tissues including testis, epidermis, wall of the aorta, as well as in subepidermal structures and mesenchymal tissues of limbs, branchial arches and tail.