BIOLOGICAL INTERVENTION BASED ON CELL AND GENE THERAPY TO IMPROVE MUSCLE HEALING AFTER LACERATION

2000 ◽  
Vol 04 (04) ◽  
pp. 265-277 ◽  
Author(s):  
Chang Woo Lee ◽  
Kazumasa Fukushima ◽  
Arvydas Usas ◽  
Lin Xin ◽  
Dalip Pelinkovic ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Functional Recovery ◽  
Sports Injuries ◽  
Ex Vivo ◽  
Healing Process ◽  
Mdx Mice ◽  
Muscle Injuries ◽  
Post Injury ◽  
Cell And Gene Therapy

Muscle laceration is a challenging problem in traumatology and is common in sports injuries, with functional recovery remaining slow and incomplete. Even though muscles retain their ability to regenerate after injury, muscles' healing process after such injuries has been found to be very slow and often leads to incomplete muscle recovery. Growth factors may have a role in enhancing recovery. Our previous study showed that IGF-1, β-FGF and NGF can improve myoblast proliferation and differentiation in vitro. We then investigated whether the delivery of IGF-1 would improve muscle healing after injuries. We observed that muscle regeneration was enhanced in lacerated muscles treated with IGF-1 protein, which consequently led to an improvement in muscle healing. However, the rapid clearance and short biological half-lives of these proteins may have limited the success of this approach. We then investigated the efficiency of gene therapy based on adenovirus to deliver a stable expression of the growth factor IGF-1. Although a slight improvement in the healing process occurred in the muscle injected with adenovirus (AIGF), the combination of myoblast transplantation and gene therapy with the ex vivo approach further improved the healing process. The injection of normal myoblasts into the injured muscle led to the best improvement of muscle healing at two weeks post-injection. Implantation of normal minced muscle into mdx mice was also capable of improving muscle healing at 2–4 weeks post-implantation. These studies will further our understanding of muscle healing post-injury and help in the development of strategies to promote efficient muscle healing and complete functional recovery after common muscle injuries.

scholarly journals Treatment of Skeletal Muscle Injury: A Review

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
L. Baoge ◽  
E. Van Den Steen ◽  
S. Rimbaut ◽  
N. Philips ◽  
E. Witvrouw ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Growth Factors ◽  
Functional Recovery ◽  
Sports Medicine ◽  
Healing Process ◽  
Muscle Injuries ◽  
Inflammatory Phase ◽  
Tgf Β1

Skeletal muscle injuries are the most common sports-related injuries and present a challenge in primary care and sports medicine. Most types of muscle injuries would follow three stages: the acute inflammatory and degenerative phase, the repair phase and the remodeling phase. Present conservative treatment includes RICE (rest, ice, compression, elevation), nonsteroidal anti-inflammatory drugs (NSAIDs) and physical therapy. However, if use improper, NSAIDs may suppress an essential inflammatory phase in the healing of injured skeletal muscle. Furthermore, it remains controversial whether or not they have adverse effects on the healing process or on the tensile strength. However, several growth factors might promote the regeneration of injured skeletal muscle, many novel treatments have involved on enhancing complete functional recovery. Exogenous growth factors have been shown to regulate satellite cell proliferation, differentiation and fusion in myotubes in vivo and in vitro, TGF-β1 antagonists behave as inhibitors of TGF-β1. They prevent collagen deposition and block formation of muscle fibrosis, so that a complete functional recovery can be achieved.

Antifibrotic effects of suramin in injured skeletal muscle after laceration

2003 ◽  
Vol 95 (2) ◽  
pp. 771-780 ◽  
Author(s):  
Yi-Sheng Chan ◽  
Yong Li ◽  
William Foster ◽  
Takashi Horaguchi ◽  
George Somogyi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Growth Factor ◽  
Muscle Regeneration ◽  
Sports Medicine ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Healing Process ◽  
Muscle Injuries

Muscle injuries are very common in traumatology and sports medicine. Although muscle tissue can regenerate postinjury, the healing process is slow and often incomplete; complete recovery after skeletal muscle injury is hindered by fibrosis. Our studies have shown that decreased fibrosis could improve muscle healing. Suramin has been found to inhibit transforming growth factor (TGF)-β1 expression by competitively binding to the growth factor receptor. We conducted a series of tests to determine the antifibrotic effects of suramin on muscle laceration injuries. Our results demonstrate that suramin (50 μg/ml) can effectively decrease fibroblast proliferation and fibrotic-protein expression (α-smooth muscle actin) in vitro. In vivo, direct injection of suramin (2.5 mg) into injured murine muscle resulted in effective inhibition of muscle fibrosis and enhanced muscle regeneration, which led to efficient functional muscle recovery. These results support our hypothesis that prevention of fibrosis could enhance muscle regeneration, thereby facilitating more efficient muscle healing. This study could significantly contribute to the development of strategies to promote efficient muscle healing and functional recovery.

scholarly journals Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5095-5103 ◽  
Author(s):  
G Hortelano ◽  
A Al-Hendy ◽  
FA Ofosu ◽  
PL Chang
Keyword(s):  
Gene Therapy ◽  
Human Factor ◽  
Ex Vivo ◽  
Cost Effective ◽  
Factor Ix ◽  
Hemophilia B ◽  
Therapy Strategy ◽  
Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

Therapeutic Effect of HOXB4-Expanded Stem Cells in Mice with Beta Thalassemia Given a Non-Myeloablative Conditioning Regimen.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 341-341
Author(s):  
Silvia Bakovic ◽  
Patricia M. Rosten ◽  
Connie J. Eaves ◽  
R. Keith Humphries
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Ex Vivo ◽  
Globin Gene ◽  
Conditioning Regimen ◽  
Beta Thalassemia ◽  
Mouse Bone Marrow ◽  
Myeloablative Conditioning ◽  
Hematopoietic Stem

Abstract The ultimate promise of gene therapy for patients with hemoglobinopathies depends on the development of safe strategies for achieving 2 goals. One is to obtain efficient and permanent correction of the gene defect in autologous hematopoietic stem cells (HSCs). The second is to develop methods for the pre-transplant amplification of transduced HSCs to high levels to ensure that they will outcompete the large residual endogenous HSC population remaining in non-myeloablated hosts (e.g. previous experiments have shown that a minimum of ~5 × 106 normal adult mouse bone marrow (BM) cells (~500 HSC) is required to achieve a level of chimerism of 20% in mice given 200 cGy). The ability of HOXB4 to promote HSC self-renewal divisions in short term culture prior to their use as transplants offers an attractive approach to achieve this latter goal. As a first test we transduced day-4 5FU BM cells from normal mice with a MSCV-HOXB4-IRES-GFP or control MSCV-IRES-GFP virus and then transplanted the cells either before or after 7 days maintenance in vitro into normal recipients given 250 cGy. Mice transplanted with an estimated 50 HSCs immediately after transduction with either virus reached equivalent low levels of chimerism (~10%) showing that HOXB4 does not impart an in vivo selective growth advantage under sublethal conditions. After ex vivo culture, the GFP transduced cells yielded an even lower level of chimerism (~5%), in contrast recipients of cultured HOXB4-transduced cells attained much higher stable levels of lympho-myeloid chimerism (~50%), indicative of a marked expansion of the HSCs pre-transplant and their retention of robust competitive repopulating potential. We then applied this approach to a gene therapy model of severe β-thalassemia in mice bearing a homozygous deletion of the β-major globin gene (β-MDD). To model a transplant of genetically corrected cells, BM cells were harvested from day-4 5FU pre-treated congenic wild-type donors and transduced with the HOXB4 virus. Cells were then cultured for 10 days and the progeny of 200K starting cells transplanted into 3 β-MDD and 4 normal recipients given 200 cGy. Transplantation of 500K freshly harvested day-4 5FU BM cells into 4 similarly conditioned control mice failed to produce significant chimerism (1–3% at 5 months). In contrast, all 4 control recipients of ex vivo expanded HOXB4-transduced cells exhibited significant stable chimerism (21±6% at 5 months). Similar levels of chimerism were also achieved in all 3 β-MDD recipients (18–76%), one of which was sustained at 34% at 5 months (52% in the RBCs). This was associated with substantial improvement in the Hct (36% vs 23% in untreated β-MDD), Hb (10.5 vs 5 g/dl) and RBC morphology. Southern blot analyses performed on 53 individual in vitro-expanded myeloid colonies generated from FACS-selected GFP+ marrow cells from this mouse 2 months post-transplant showed 19 distinct integration patterns indicating reconstitution from polyclonal expanded HSCs. This conclusion was further confirmed by proviral integration site analyses, which identified 13 separate integration sites from 9 colonies that had unique proviral patterns. These data demonstrate the curative potential of ex vivo expanded HSCs in a preclinical model of β-thalassemia treated with non-myeloablative conditioning. They also underscore the potential of HOXB4 as a potent tool to achieve the HSC expansions required.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5143-5143
Author(s):  
Liesbeth De Waele ◽  
Kathleen Freson ◽  
Chantal Thys ◽  
Christel Van Geet ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Phosphoglycerate Kinase ◽  
Lentiviral Vectors ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Platelet Disorders

Abstract The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages. Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

Autologous, Orthotopic Thyroid Follicular Cell Transplantation: A Surgical Component of Ex Vivo Somatic Gene Therapy

1993 ◽  
Vol 108 (1) ◽  
pp. 51-62 ◽  
Author(s):  
Bert W. O'Malley ◽  
Milton J. Finegold ◽  
Fred D. Ledley
Keyword(s):  
Gene Therapy ◽  
Ex Vivo ◽  
Genetic Material ◽  
Genetically Engineered ◽  
Follicular Cells ◽  
Somatic Gene Therapy ◽  
Orthotopic Transplantation ◽  
Somatic Gene ◽  
Thyroid Follicular Cells

Ex vivo strategies for somatic gene therapy involve the harvest of primary cells from patients, the transfer of novel genetic material into these cells in cell culture, and reimplantation of the genetically engineered cells back into patients. In consideration of methods for targeting somatic gene therapy to the thyroid, we have studied the autologous, orthotopic transplantation of thyroid follicular cells in a canine model. Using the fluorescent dye Dil, we were able to stain follicular cells in vitro before transplantation and then follow the pattern of engraftment through histologic sectioning and microscopy up to 14 days after transplantation. The transplantations involved the direct injection of intact and disrupted follicles into a remaining thyroid lobe after cell harvest from the contralateral lobe. We also demonstrated engraftment of individual follicular cells recovered from primary monolayer cultures. Histologic studies revealed the presence of transplanted cells and follicles as well as focal regions of granulomatous reaction in close relation to the engrafted material. These studies demonstrate the feasibility of autologous, orthotopic transplantation of thyroid follicular cells. This method is an essential component of ex vivo strategies for targeting somatic gene therapy to the thyroid gland.

The Effect of Relaxin Treatment on Skeletal Muscle Injuries

2005 ◽  
Vol 33 (12) ◽  
pp. 1816-1824 ◽  
Author(s):  
Shinichi Negishi ◽  
Yong Li ◽  
Arvydas Usas ◽  
Freddie H. Fu ◽  
Johnny Huard
Keyword(s):  
Skeletal Muscle ◽  
Growth Factor ◽  
Functional Recovery ◽  
Muscle Regeneration ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
C2c12 Cells ◽  
Muscle Injuries

Background Injured skeletal muscle can repair itself via spontaneous regeneration; however, the overproduction of extracellular matrix and excessive collagen deposition lead to fibrosis. Neutralization of the effect of transforming growth factor-β1, a key fibrotic cytokine, on myogenic cell differentiation after muscle injury can prevent fibrosis, enhance muscle regeneration, and thereby improve the functional recovery of injured muscle. Hypothesis The hormone relaxin, a member of the family of insulin-like growth factors, can act as an antifibrosis agent and improve the healing of injured muscle. Study Design Controlled laboratory study. Methods In vitro: Myoblasts (C2C12 cells) and myofibroblasts (transforming growth factor-β1-transfected myoblasts) were incubated with relaxin, and cell growth and differentiation were examined. Myogenic and fibrotic protein expression was determined by Western blot analysis. In vivo: Relaxin was injected intramuscularly at different time points after laceration injury. Skeletal muscle healing was evaluated via histologic, immunohistochemical, and physiologic tests. Results Relaxin treatment resulted in a dose-dependent decrease in myofibroblast proliferation, down-regulated expression of the fibrotic protein α-smooth muscle actin, and promoted the proliferation and differentiation of myoblasts in vitro. Relaxin therapy enhanced muscle regeneration, reduced fibrosis, and improved injured muscle strength in vivo. Conclusion Administration of relaxin can significantly improve skeletal muscle healing. Clinical Relevance These findings may facilitate the development of techniques to eliminate fibrosis, enhance muscle regeneration, and improve functional recovery after muscle injuries.

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