Rapid generation of Col7a1-/- mouse model of recessive dystrophic epidermolysis bullosa and partial rescue via immunosuppressive dermal mesenchymal stem cells.

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating and ultimately lethal blistering disease caused by mutations to the Col7a1 gene. Development of novel cell therapies for the treatment of RDEB would be fostered by having immunodeficient mouse models able to accept human cell grafts; however, immunodeficient models of many genodermatoses such as RDEB are lacking. To overcome this limitation, we combined the clustered regularly interspaced short palindromic repeats and associated nuclease (CRISPR/Cas9) system with microinjection into NOD/SCID IL2rγcnull (NSG) embryos to rapidly develop an immunodeficient Col7a1-/- mouse model of RDEB. Through dose optimization, we achieve F0 biallelic knockout efficiencies exceeding 80%, allowing us to quickly generate large numbers of RDEB NSG mice for experimental use. Using this strategy, we clearly demonstrate important strain-specific differences in RDEB pathology that could underlie discordant results observed between independent studies and establish the utility of this system in proof-of-concept human cellular transplantation experiments. Importantly, we uncover the ability of a recently identified skin resident immunomodulatory dermal mesenchymal stem cell marked by ABCB5 to reduce RDEB pathology and markedly extend the lifespan of RDEB NSG mice via reduced skin infiltration of inflammatory myeloid derivatives.

Lentivirus transduction of human osteoclast precursor cells and differentiation into functional osteoclasts.

Gene transfer into stem cells has been an ongoing priority as a treatment for genetic disease and cancer for more than two decades. Methods described herein, form the basis for providing the cell source to determine if osteoclast precursor cells (OcP) can be used as therapeutic gene delivery systems in vivo. Osteoclasts and tumor associated macrophages or OcP, support survival, tumor progression and osteolysis in bone cancers. Two sources of precursor cells are compared: CD14+ cells, the standard OcP, found abundantly in peripheral blood and CD34+ cells, hematopoietic stem cells that are rare, but which can be expanded into OcP. Our findings characterize cell yield at each step of the transduction process and thus provide essential data for planning future in vivo experiments. In addition we demonstrate that essential functions of OcP are preserved following lentiviral transduction. Specifically, neither the transduction method nor the lentiviral transduction influence the OcP's ability to form osteoclasts, express the marker gene, EGFP, or resorb bone. Finally, we conclude that CD34+ cells yield significantly more transduced cells and form functionally superior osteoclasts in vitro. This study represents a step towards considering human gene therapy for bone cancer by demonstrating successful transduction of human OcP for use as cellular delivery vehicles to sites of bone cancer.

Osteoclasts direct bystander killing of bone cancer.

Primary and metastatic bone cancers are difficult to eradicate and novel approaches are needed to improve treatment and extend life. As bone cancer grows, osteoclasts, the principal bone-resorbing cells of the body, are recruited to and activated at sites of cancer. In this investigation, we determined if osteoclast lineage cells could function as a cell-based gene delivery system to bone cancers. We used the cytosine deaminase (CD) 5-fluorocytosine (5-FC) enzyme/prodrug system and studied bone marrow and bones from transgenic mice expressing a novel CD gene regulated by the osteoclast tartrate-resistant acid phosphatase (TRAP) gene promoter (Tg/NCD). DsRed2-labeled 2472 sarcoma cells were placed in Tg/NCD osteoclastogenic cultures and treated with 5-FC. 5-FC treatment resulted in profound bystander killing (90%; P < 0.05). The effect of 5-FC treatment on osteoclast lineage cells was most dramatic when administered at the beginning of the 7-day cultures, suggesting that mature osteoclasts are less sensitive to 5-FC. Evaluation of osteoclast-directed bystander killing in vivo revealed dramatic killing of bone cancer with only a modest effect on osteoclast number. Specifically, 5-FC treatment of tumor-bearing Tg/NCD mice or Tg/NCD bone marrow transplanted C3H mice (Tg/NCD-C3H) resulted in 92% and 44% reductions in tumor area, respectively (P < 0.05). Eight of ten 5-FC-treated Tg/NCD mice had complete bone tumor killing and five of six 5-FC-treated Tg/NCD-C3H mice had reduced tumor compared with controls. In addition, Tg/NCD osteoclasts were resistant to 5-FC treatment in vivo, a very important feature, as it identifies osteoclasts as an ideal CD gene delivery system.