The host range of retroviral vectors including lentiviral vectors can be expanded or altered by a process known as pseudotyping. Pseudotyped lentiviral vectors consist of vector particles bearing glycoproteins (GPs) derived from other enveloped viruses. Such particles possess the tropism of the virus from which the GP was derived. For example, to exploit the natural neural tropism of rabies virus, vectors designed to target the central nervous system have been pseudotyped using rabies virusderived GPs. Among the first and still most widely used GPs for pseudotyping lentiviral vectors is the vesicular stomatitis virus GP (VSV-G), due to the very broad tropism and stability of the resulting pseudotypes. Pseudotypes involving VSV-G have become effectively the standard for evaluating the efficiency of other pseudotypes. This review samples a few of the more prominent examples from the ever-expanding list of published lentiviral pseudotypes, noting comparisons made with pseudotypes involving VSV-G in terms of titer, viral particle stability, toxicity, and host-cell specificity. Particular attention is paid to publications of successfully targeting a specific organ or cell types.

Bone marrow-derived mesenchymal stromal cells (MSCs) have attracted attention as potential platforms for the systemic delivery of therapeutic proteins in vivo following gene transfer using oncogenic retroviruses. However, the major limitations of this strategy include low levels of gene transfer and a general lack of long-term transgene expression. We have investigated the expression of several transgenes in MSCs following HIV-1 lentiviral vector-mediated gene transfer. Vectors containing a variety of strong promoters driving enhanced green fluorescence protein (EGFP) and coral (Discosoma sp.)-derived red fluorescent protein (DsRed) reporter genes pseudotyped with the vesicular stomatitis virus-G (VSV-G) glycoprotein were able to transduce cultured MSCs with high efficiency. Transduction efficiencies and transgene expression levels in MSCs were found to be higher with lentiviral vectors than with a vector based on the murine stem cell virus pseudotyped with VSV-G. Transgene expression was maintained in culture for at least 5 months. HIV-1-based lentiviral vectors were able to transduce clonogenic mesenchymal progenitor cells, which were capable of maintaining transgene expression by their MSC progeny, over several cell divisions and during differentiation into adipocytes, indicating that terminal adipocyte cell differentiation was unaffected by lentivirus-mediated reporter gene transfer. Collectively these results suggest that lentivirus-mediated gene transfer strategies provide an efficient tool for ex vivo modification of MSCs that does not interfere with differentiation.

The intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells, including the pluripotent haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and possibly the multipotent adherent progenitor cells (MAPCs), are being considered as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineagesn and, at least in vitro, have significant expansion capability. The apparently high self-renewal potential makes them strong candidates for delivering genes and restoring organ systems function. However, the high proliferative potential of MSCs, now presumed to be self-renewal, may be more apparent than real. Although expanded MSCs have great proliferation and differentiation potential in vitro, there are limitations with the biology of these cells in vivo. So far, expanded MSCs have failed to induce durable therapeutic effects expected from a true self-renewing stem cell population. The loss of in vivo self-renewal may be due to the extensive expansion of MSCs in existing in vitro expansion systems, suggesting that the original stem cell population and/or properties may no longer exist. Rather, the expanded population may indeed be heterogeneous and represents several generations of different types of mesenchymal cell progeny that have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages. Novel technology that allows MSCs to maintain their stem cell function in vivo is critical for distinguishing the elusive stem cell from its progenitor cell populations. The ultimate dream is to use MSCs in various forms of cellular therapies, as well as genetic tools that can be used to better understand the mechanisms leading to repair and regeneration of damaged or diseased tissues and organs.

No abstract

A number of studies have shown that a short peptide, the protein transduction domain (PTD) derived from the HIV‐1 Tat protein (Tat‐PTD) improved cellular uptake in vitro and distribution in vivo of recombinant proteins bearing such PTDs when administered systemically. To investigate the effects of Tat‐PTD addition on the subcellular localization of the lysosomal enzyme galactocerebrosidase (GALC, EC 3.2.2.46) and with a view towards designing improved therapeutic strategies for Krabbe disease (globoid cell leukodystrophy), mouse GALC was tagged C‐terminally with the Tat‐PTD. Compared with unmodified GALC, GALC bearing a Tat‐PTD, a myc epitope and 6 consecutive His residues [GALC‐TMH (Tat‐PTD, a myc epitope and 6 consecutive His residues)] was found to be secreted more efficiently. Also, GALC‐TMH was found to be taken up by cells both via mannose‐6‐phosphate receptor (M6PR)‐mediated endocytosis as well as by M6PR‐independent mechanisms. GALC‐TMH displayed increased M6PR‐independent uptake in fibroblasts derived from twitcher mice (a murine model of globoid cell leukodystrophy) and in neurons derived from the mouse brain cortex compared with GALC lacking a Tat‐PTD. Immunocytochemical analyses revealed that Tat‐modified GALC protein co‐localized in part with the lysosome‐associated membrane protein‐1. Complete correction of galactosylceramide accumulation was achieved in twitcher mouse fibroblasts lacking GALC activity following addition of GALC‐TMH. Therefore, GALC‐TMH not only maintained the features of the native GALC protein including enzymatic function, intracellular transport and location, but also displayed more efficient cellular uptake.