"What Did Maxwell's Equations Really Have to Do With Edison's Invention?": Addressing the Complexity of Developing Clinical Interventions for Skeletal Muscle Disease.

To reach the healthcare market and have a medical intervention reimbursed in any format carries high risk and very low success rates. Even when all regulatory hurdles have been surpassed, there is no guarantee that the product will be purchased; a different body makes that decision using criteria typically unknown to early-stage innovators and intervention developers. In the context of skeletal muscle diseases, the field is at a crossroads; accurate diagnoses are difficult to obtain, patient management and monitoring are equally difficult, cures are evasive, and disease progression is not well enough understood in the human to identify clear targets (irrespective of whether the specific muscle disease is rare or frequent because the progression is slow and the tissue large). Additionally, the generation of fundamental knowledge stemming from pure academic research, which underpins short- and long-term insight and advances, has been stalled or at least slowed. The field also faces challenges common to all healthcare interventions, while there are also some unique barriers to address, in both developmental translation of the therapeutic and obtaining reimbursement approval. This is independent of the number of people globally who suffer directly and indirectly from skeletal muscle degeneration or degradation. This chapter covers key issues facing skeletal muscle intervention translation, problems that seem to be routinely occurring, followed by suggestions on what can and should be done differently.

Molecular purging of multiple myeloma cells by ex-vivo culture and retroviral transduction of mobilized-blood CD34+ cells.

BACKGROUND: Tumor cell contamination of the apheresis in multiple myeloma is likely to affect disease-free and overall survival after autografting. OBJECTIVE: To purge myeloma aphereses from tumor contaminants with a novel culture-based purging method. METHODS: We cultured myeloma-positive CD34+ PB samples in conditions that retained multipotency of hematopoietic stem cells, but were unfavourable to survival of plasma cells. Moreover, we exploited the resistance of myeloma plasma cells to retroviral transduction by targeting the hematopoietic CD34+ cell population with a retroviral vector carrying a selectable marker (the truncated form of the human receptor for nerve growth factor, DeltaNGFR). We performed therefore a further myeloma purging step by selecting the transduced cells at the end of the culture. RESULTS: Overall recovery of CD34+ cells after culture was 128.5%; DeltaNGFR transduction rate was 28.8% for CD34+ cells and 0% for CD138-selected primary myeloma cells, respectively. Recovery of CD34+ cells after DeltaNGFR selection was 22.3%. By patient-specific Ig-gene rearrangements, we assessed a decrease of 0.7-1.4 logs in tumor load after the CD34+ cell selection, and up to 2.3 logs after culture and DeltaNGFR selection. CONCLUSION: We conclude that ex-vivo culture and retroviral-mediated transduction of myeloma leukaphereses provide an efficient tumor cell purging.

Mobilized blood CD34+ cells transduced and selected with a clinically applicable protocol reconstitute lymphopoiesis in SCID-Hu mice.

We developed a clinically applicable gene transfer procedure into mobilized peripheral blood (MPB) CD34(+) hematopoietic progenitor cells, based on single viral exposure and selection of engineered cells. CD34(+) cells were transduced with a retroviral vector carrying the truncated form of the nerve growth factor receptor (Delta NGFR) marker gene, and immunoselected for Delta NGFR expression. Optimal time and procedure for viral exposure, length of culture, and transgene expression of MPB CD34(+) cells were determined using in vitro assays. The multipotent capacity of MPB CD34(+)-transduced cells was demonstrated in the SCID-hu bone/liver/thymus mouse model. Transduced Delta NGFR(+) cells retained 50% of long-term culture-colony forming cells (LTC-CFC) compared to unmanipulated CD34(+) cells. In SCID-hu mice, 52% of CD45(+) cells, 27% of CD34(+) cells, 49% of B cells, and more than 50% of T cells were derived from transplanted CD34(+)/Delta NGFR(+) cells. Furthermore, transplantation of purified transduced cells greatly reduced the competition with untransduced progenitors occurring in unselected grafts. These data demonstrate that MPB CD34(+) cells, transduced with a single viral exposure and selected by transgene expression, retain multilineage reconstitution capacity and remarkable transgene expression.

Efficient gene transfer into primitive hematopoietic progenitors using a bone marrow microenvironment cell line engineered to produce retroviral vectors.

BACKGROUND AND OBJECTIVES: Effective gene transfer into human hematopoietic stem/progenitor cells is a compromise between achieving high transduction efficiency and maintaining the desired biological characteristics of the target cell. The aim of our work was to exploit the stromal microenvironment to increase gene transfer and maintenance of hematopoietic progenitors. DESIGN AND METHODS: The murine bone marrow stromal cell line MS-5, known to support primitive human progenitors, was modified into an amphotropic packaging cell, by the stable introduction of DNA coding for retroviral structural proteins, and a viral vector encoding a marker gene. The gene transfer efficiency of the recombinant virus was evaluated by flow cytometry, in vitro assays for committed (CFC) and primitive (LTC-CFC) progenitors, as well as a clonal assay for B and NK lymphoid progenitors. RESULTS: The new packaging cell line (NEXUS) produced equivalent levels of virus as did the established GP+Am12 system, also under serum-free conditions. On average 30% of human mobilized peripheral blood CD34(+) cells were transduced by a single exposure to NEXUS supernatant, representing a three-fold increase over GP+Am12-based technology. Gene transfer into both committed and primitive progenitors increased on average two-fold using NEXUS retroviral supernatant. Furthermore, CD34(+) CD38(low) early progenitor cells purified from umbilical cord blood were efficiently transduced with NEXUS retroviral vector and gave rise to a high frequency of marked B and NK lymphocytes. INTERPRETATION AND CONCLUSIONS: Our data show that that an established bone marrow stromal cell can be engineered to enhance the genetic modification of primitive hematopoietic and lymphoid progenitors using a clinically relevant method.

Notch/Delta4 interaction in human embryonic liver CD34+ CD38- cells: positive influence on BFU-E production and LTC-IC potential maintenance.

We investigated whether Notch signaling pathways have a role in human developmental hematopoiesis. In situ histochemistry analysis revealed that Notch1, 2, and 4 and Notch ligand (Delta1-4, and Jagged1) proteins were not expressed in the yolk sac blood islands, the para-aortic splanchnopleure, the hematopoietic aortic clusters, and at the early stages of embryonic liver hematopoiesis. Notch1-2, and Delta4 were eventually detected in the embryonic liver, from 34 until 38 days postconception. Fluorescence-activated cell sorter analysis showed that first-trimester embryonic liver CD34(+)CD38(low) cells expressed both Notch1 and Notch2. When these cells were cultured on S17 stroma stably expressing Delta4, a 2.6-fold increase in BFU-E number was observed at day 7, as compared with cultures with control stroma, and this effect was maintained for 2 weeks. Importantly, exposure of these cells to Delta4 under these conditions maintained the original frequency and quality of long-term culture-initiating cells (LTC-ICs), while control cultures quickly resulted in the extinction of this LTC-IC potential. Furthermore, short-term exposure of embryonic liver adherent cells to erythropoietin resulted in a dose-dependent increase in Delta4 expression, almost doubling the expression observed with untreated stroma. This suggests that Delta4 has a role in the regulation of hematopoiesis after a hypoxic stress in the fetus.