scholarly journals Keratinocyte Gene Transfer and Gene Therapy

1996 ◽  
Vol 7 (3) ◽  
pp. 204-221 ◽  
Author(s):  
J.A. Garlick ◽  
E.S. Fenjves
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Target Cells ◽  
Gene Products ◽  
Oral Keratinocytes ◽  
Systemic Delivery ◽  
Therapy Strategies

Gene therapy has moved beyond the pre-clinical stage to the treatment of a variety of inherited and acquired diseases. For such therapy to be successful, genes must be efficiently delivered to target cells and gene products must be expressed for prolonged periods of time without toxic effects to the host. This may be achieved by means of an in vivo strategy where genes are transferred directly into a host cell, or by means of an ex vivo approach through which cells are removed, cultured, targeted for gene delivery, and grafted back to the host. Several obstacles continue to delay safe and effective clinical application of gene therapy in a variety of target cells. The limited survival of transplanted cells, transient expression of transferred genes, and difficulties in targeting stem cells are technical issues requiring further investigation. Epidermal and oral keratinocytes are potential vehicles for gene therapy. Several features of these tissues can be utilized to achieve delivery of therapeutic gene products for local or systemic delivery. These qualities include: (1) the presence of stem cells; (2) the cell-, strata-, and site-specific regulation of keratinocyte gene expression; (3) tissue accessibility; and (4) secretory capacity. Such features can be exploited by the use of gene therapy strategies to facilitate: (1) identification, enrichment, and targeting of stem cells to ensure the continued presence of the transferred gene; (2) high-level and persistent transgene expression using keratinocyte-specific promoters; (3) tissue access needed for culture and grafting for ex vivo therapy and direct in vivo gene transfer; (4) secretion of transgene product for local or systemic delivery; and (5) monitoring of genetically modified tissue and removal if treatment termination is required. Optimal gene therapy strategies are being tested in a variety of tissues to treat dominant and recessive genetic disorders as well as acquired diseases such as neoplasia and infectious disease. This experience provides a basis for the application of such clinical studies to a spectrum of diseases effecting epidermal and oral keratinocytes. Gene therapy is in an early stage yet holds great promise for its ultimate clinical application.

scholarly journals Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model

2021 ◽  
Vol 18 ◽  
pp. 347-354
Author(s):  
Masashi Noda ◽  
Kohei Tatsumi ◽  
Hideto Matsui ◽  
Yasunori Matsunari ◽  
Takeshi Sato ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Canine Model ◽  
Gene Transfer Techniques

scholarly journals Transduction of Bone-Marrow-Derived Mesenchymal Stem Cells by Using Lentivirus Vectors Pseudotyped with Modified RD114 Envelope Glycoproteins

2004 ◽  
Vol 78 (3) ◽  
pp. 1219-1229 ◽  
Author(s):  
Xian-Yang Zhang ◽  
Vincent F. La Russa ◽  
Jakob Reiser
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Leukemia Virus ◽  
Human Mscs ◽  
Systemic Delivery ◽  
Endogenous Virus ◽  
Hiv 1

ABSTRACT Bone-marrow-derived mesenchymal stem cells (MSCs) have attracted considerable attention as tools for the systemic delivery of therapeutic proteins in vivo, and the ability to efficiently transfer genes of interest into such cells would create a number of therapeutic opportunities. We have designed and tested a series of human immunodeficiency virus type 1 (HIV-1)-based vectors and vectors based on the oncogenic murine stem cell virus to deliver and express transgenes in human MSCs. These vectors were pseudotyped with either the vesicular stomatitis virus G (VSV-G) glycoprotein (GP) or the feline endogenous virus RD114 envelope GP. Transduction efficiencies and transgene expression levels in MSCs were analyzed by quantitative flow cytometry and quantitative real-time PCR. While transduction efficiencies with virus particles pseudotyped with the VSV-G GP were found to be high, RD114 pseudotypes revealed transduction efficiencies that were 1 to 2 orders of magnitude below those observed with VSV-G pseudotypes. However, chimeric RD114 GPs, with the transmembrane and extracellular domains fused to the cytoplasmic domain derived from the amphotropic Moloney murine leukemia virus 4070A GP, revealed about 15-fold higher titers relative to the unmodified RD114 GP. The transduction efficiencies in human MSCs of HIV-1-based vectors pseudotyped with the chimeric RD114 GP were similar to those obtained with HIV-1 vectors pseudotyped with the VSV-G GP. Our results also indicate that RD114 pseudotypes were less toxic than VSV-G pseudotypes in human MSC progenitor assays. Taken together, these results suggest that lentivirus pseudotypes bearing alternative Env GPs provide efficient tools for ex vivo modification of human MSCs.

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.

scholarly journals Ex Vivo and In Vivo Adenovirus-Mediated Gene Therapy Strategies Induce a Systemic Anti-Tumor Immune Defence in the B16 Melanoma Model

1998 ◽  
Vol 110 (6) ◽  
pp. 867-871 ◽  
Author(s):  
Bernd Bonnekoh ◽  
David A. Greenhalgh ◽  
Shu-Hsia Chen ◽  
Andreas Block ◽  
Susan S. Rich ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Ex Vivo ◽  
B16 Melanoma ◽  
Immune Defence ◽  
Melanoma Model ◽  
Therapy Strategies

scholarly journals Efficient and Highly Specific Gene Transfer Using Mutated Lentiviral Vectors Redirected with Bispecific Antibodies

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christina L. Parker ◽  
Timothy M. Jacobs ◽  
Justin T. Huckaby ◽  
Dimple Harit ◽  
Samuel K. Lai
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Endosomal Escape ◽  
Bispecific Antibodies ◽  
Target Cells ◽  
Specific Gene ◽  
Targeted Gene Delivery

ABSTRACT Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo. We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2+) (SKBR3) breast and HER2-negative (HER2−) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy. IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo, expanding the current use of LV beyond ex vivo applications.

Emerging potential of transposons for gene therapy and generation of induced pluripotent stem cells

Blood ◽  
2009 ◽  
Vol 114 (8) ◽  
pp. 1461-1468 ◽  
Author(s):  
Thierry VandenDriessche ◽  
Zoltán Ivics ◽  
Zsuzsanna Izsvák ◽  
Marinee K. L. Chuah
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
B Cell Lymphoma ◽  
Sleeping Beauty ◽  
Target Cells ◽  
Stable Gene ◽  
Induced Pluripotent

AbstractEffective gene therapy requires robust delivery of the desired genes into the relevant target cells, long-term gene expression, and minimal risks of secondary effects. The development of efficient and safe nonviral vectors would greatly facilitate clinical gene therapy studies. However, nonviral gene transfer approaches typically result in only limited stable gene transfer efficiencies in most primary cells. The use of nonviral gene delivery approaches in conjunction with the latest generation transposon technology based on Sleeping Beauty (SB) or piggyBac transposons may potentially overcome some of these limitations. In particular, a large-scale genetic screen in mammalian cells yielded a novel hyperactive SB transposase, resulting in robust and stable gene marking in vivo after hematopoietic reconstitution with CD34+ hematopoietic stem/progenitor cells in mouse models. Moreover, the first-in-man clinical trial has recently been approved to use redirected T cells engineered with SB for gene therapy of B-cell lymphoma. Finally, induced pluripotent stem cells could be generated after genetic reprogramming with piggyBac transposons encoding reprogramming factors. These recent developments underscore the emerging potential of transposons in gene therapy applications and induced pluripotent stem generation for regenerative medicine.

Nonviral gene therapy targeting cardiovascular system

2012 ◽  
Vol 303 (6) ◽  
pp. H629-H638 ◽  
Author(s):  
Cheng-Huang Su ◽  
Yih-Jer Wu ◽  
Hsueh-Hsiao Wang ◽  
Hung-I Yeh
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Potential Method ◽  
Therapeutic Angiogenesis ◽  
Advanced Technology ◽  
Target Cells ◽  
Peripheral Tissues

The goal of gene therapy is either to introduce a therapeutic gene into or replace a defective gene in an individual's cells and tissues. Gene therapy has been urged as a potential method to induce therapeutic angiogenesis in ischemic myocardium and peripheral tissues after extensive investigation in recent preclinical and clinical studies. A successful gene therapy mainly relies on the development of the gene delivery vector. Developments in viral and nonviral vector technology including cell-based gene transfer will further improve transgene delivery and expression efficiency. Nonviral approaches as alternative gene delivery vehicles to viral vectors have received significant attention. Recently, a simple and safe approach of gene delivery into target cells using naked DNA has been improved by combining several techniques. Among the physical approaches, ultrasonic microbubble gene delivery, with its high safety profile, low costs, and repeatable applicability, can increase the permeability of cell membrane to macromolecules such as plasmid DNA by its bioeffects and can provide as a feasible tool in gene delivery. On the other hand, among the promising areas for gene therapy in acquired diseases, ischemic cardiovascular diseases have been widely studied. As a result, gene therapy using advanced technology may play an important role in this regard. The aims of this review focus on understanding the cellular and in vivo barriers in gene transfer and provide an overview of currently used chemical vectors and physical tools that are applied in nonviral cardiovascular gene transfer.

Adenoviral gene transfer of eNOS: high-level expression in ex vivo expanded marrow stromal cells

2003 ◽  
Vol 285 (5) ◽  
pp. C1322-C1329 ◽  
Author(s):  
Weiwen Deng ◽  
Trinity J. Bivalacqua ◽  
Natasha N. Chattergoon ◽  
Albert L. Hyman ◽  
James R. Jeter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Stromal Cells ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Adult Stem Cell ◽  
Marrow Stromal Cells ◽  
Enos Gene ◽  
High Level

Endothelial nitric oxide synthase (eNOS) is an attractive target for cardiovascular gene therapy. Marrow stromal cells (MSCs), also known as mesenchymal stem cells, hold great promise for use in adult stem cell-based cell and gene therapy. To determine the feasibility of adenoviral-mediated eNOS gene transfer into ex vivo expanded MSCs, rat MSCs (rMSCs) were isolated, expanded ex vivo, and transduced with Ad5RSVeNOS, an adenoviral vector containing the eNOS gene under the control of the Rous sarcoma virus promoter. The presence of eNOS protein in Ad5RSVeNOS-transduced rMSCs was confirmed by immunohistochemical and Western blot analysis. Transduction efficiency was dose dependent, and eNOS transgene expression in rMSCs persisted for ≥21 days in culture. The rMSCs retained multipotential differentiation capability after adenoviral-mediated eNOS gene transfer. Furthermore, intracavernosal injection of Ad5RSVeNOS-transduced rMSCs increased the expression of eNOS in the corpus cavernosum, and stem cells were identified within corporal sinusoids. These findings demonstrate that replication-deficient recombinant adenovirus can be used to engineer ex vivo expanded rMSCs and that high-level eNOS transgene expression can be achieved, pointing out the clinical potential of using this novel adult stem cell-based gene therapy method for the treatment of cardiovascular diseases.

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