The skin is an attractive tissue for gene therapy applications to treat genetic disorders and to express systemically delivered transgenes encoding therapeutic proteins. Understanding the tissue tropism of vectors is a prerequisite for the design of gene therapy trials. Using an ex vivo system of organ culture, we studied factors that determined viral tropism to the epidermal and dermal cells in human and mouse skin. We applied in these studies a lentiviral vector pseudotyped with two glycoproteins that use different cell receptors (vesicular stomatitis virus glycoprotein [VSV-G] and amphotropic murine leukemia virus envelope). The extent of infection with the amphotropic pseudotype was much higher than that of VSV-G, especially at low multiplicities of infection. In contrast, the tropism of these two pseudotypes in skin tissues was similar; at low multiplicities the infection was limited to areas near the basal layer of the epidermis, whereas at high multiplicities the infection extended to the dermal layer. To overcome physical barriers in the skin, the epidermal and dermal layers were separated and infected. Whereas the human epidermis was readily infected, we could not detect infection of stem and early progenitor cells in their niche. In contrast, mouse epidermis was completely resistant to infection. Dermal cells of both species were readily infected with the two pseudotypes. Molecular analysis indicated that infection of mouse epidermal cells was restricted after proviral DNA synthesis and before integration. In conclusion, we show that lentiviral tropism in a solid tissue is dependent on several factors, extra- and intracellular, distinct of the cellular receptors.

Taken together, the results obtained in the present study indicate that lentiviral vector tropism in the three-dimensional skin tissue is distinct from the tropism to keratinocytes in culture and is dependent on a complex interplay of extracellular restrictions.

A novel approach for sustained production of therapeutic proteins is described, using genetic modification of intact autologous micro-organ tissue explants from the subject's own skin. The skin-derived micro-organ can be maintained viable ex vivo for extended periods and is transduced with a transgene encoding a desired therapeutic protein, resulting in protein-secreting micro-organ (biopump (BP)). The daily protein production from each BP is quantified, enabling drug dosing by subcutaneous implantation of the requisite number of BPs into the patient to provide continuous production to the circulation of a known amount of the therapeutic protein. Each implanted BP remains localized and is accessible, to enable removal or ablation if needed. Examples from preclinical and clinical studies are presented, including use of associated virus vector 1 and helper-dependent adenoviral vectors producing BPs to provide long-term sustained secretion of recombinant interferon-α and erythropoietin.

The results indicated a specific pattern of viral tropism to skin cancer cells that are critical for maintenance of the tumour. This new experimental system should aid in the analysis of new therapeutic modalities, such as oncolytic viruses, for future treatment of these skin tumours.

The whole world has been affected by a dramatically increasing prevalence of diabetes. Today, the etiology of both type 1 and type 2 diabetes is thought to revolve around the dysfunction of β-cells, the insulin producing cells of the body. Within the pharmaceutical industry, the evaluation of new drugs for diabetes treatment is mostly done using cell lines or rodent islets and depends solely on the assessment of static insulin secretion. However, the use of cell lines or rodent islets is limiting lack of similarity of the human islet cells, leading to a constrain of the predictive value regarding the clinical potential of newly developed drugs. To overcome this issue, we developed an Engineered Micro-Pancreas as a unique platform for drug discovery. The Engineered Micro Pancreas is composed of (i) an organ-derived micro-scaffold, specifically a decellularized porcine lung-derived micro-scaffold and (ii) cadaveric islets seeded thereon. The Engineered Micro Pancreas remained viable and maintained insulin secretion in vitro for up to three months. The quantities of insulin were comparable to those secreted by freshly isolated human islets and therefore hold the potential for real-time and metabolic physiology mimicking drug screening.