906. Effective Repetitive Dystrophin Gene Transfer into Skeletal Muscle of Adult mdx Mice Using a Helper-Dependent Adenovirus Vector Expressing the Coxsackievirus and Adenovirus Receptor (CAR) and Dystrophin

ABSTRACT Recombinant adenoviruses (Ad) have become the vector system of choice for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to cells expressing marginal levels of the coxsackievirus and adenovirus receptor (CAR). In order to achieve CAR-independent gene transfer by Ad vectors in clinically important contexts, we proposed modification of viral tropism via genetic alterations to the viral fiber protein. We have shown that incorporation of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain results in the ability of the virus to utilize an alternative receptor during the cell entry process. We have also demonstrated that due to its expanded tissue tropism, this novel vector is capable of efficient transduction of primary tumor cells. An increase in gene transfer to ovarian cancer cells of 2 to 3 orders of magnitude was demonstrated by the vector, suggesting that recombinant Ad containing fibers with an incorporated RGD peptide may be of great utility for treatment of neoplasms characterized by deficiency of the primary Ad type 5 receptor.

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Reduction of Natural Adenovirus Tropism to the Liver by both Ablation of Fiber-Coxsackievirus and Adenovirus Receptor Interaction and Use of Replaceable Short Fiber

Journal of Virology ◽

10.1128/jvi.77.4.2512-2521.2003 ◽

2003 ◽

Vol 77 (4) ◽

pp. 2512-2521 ◽

Cited By ~ 84

Author(s):

Takafumi Nakamura ◽

Kenzo Sato ◽

Hirofumi Hamada

Keyword(s):

Gene Transfer ◽

Adenovirus Vector ◽

Short Fiber ◽

Transduction Efficiency ◽

Target Cells ◽

Shaft Length ◽

Adenovirus Serotype ◽

Coxsackievirus And Adenovirus Receptor ◽

Adenovirus Receptor

ABSTRACT The initial recognition and binding of adenovirus vector to the host cell surface is mediated by interaction between the adenovirus fiber knob protein and its receptor, the coxsackievirus and adenovirus receptor (CAR). This natural tropism of adenovirus vector needs to be ablated in order to achieve targeted gene transfer. To this end, we noted that adenovirus serotype 40 (Ad40) contains two distinct long and short fibers; the short fiber is unable to recognize CAR, while the long fiber binds CAR. We generated adenovirus serotype 5-based mutants with chimeric Ad40-derived fibers, which were composed of either long or short shafts together with CAR binding or nonbinding knobs. The capacity of these adenovirus mutants for in vitro and in vivo gene transfer to liver cells was examined. In the case of primary human hepatocytes displaying a high expression level of CAR and αv integrin, both CAR binding ability and fiber shaft length played important roles in efficient transduction. Most significantly, the high transduction efficiency observed in the liver and spleen following intravenous administration of adenovirus vector was dramatically reduced by both ablation of fiber-CAR interaction and the use of replaceable short fiber. In other tissues displaying a low level of transduction, no significant differences in transduction efficiency were observed among adenovirus vector mutants. Furthermore, incorporation of a 7-lysine-residue motif at the C-terminal end of CAR-nonbinding short fiber efficiently achieved transduction of target cells via the heparan-containing receptor. Our results demonstrated that the natural tropism of adenovirus in vivo is influenced not only by fiber-CAR interaction but also by fiber shaft length. Furthermore, our strategy may be useful for retargeting adenovirus to particular tumors and tissue types with specific receptors.

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Autophagy in the heart is enhanced and independent of disease progression in mus musculus dystrophinopathy models

JRSM Cardiovascular Disease ◽

10.1177/2048004019879581 ◽

2019 ◽

Vol 8 ◽

pp. 204800401987958

Author(s):

HR Spaulding ◽

C Ballmann ◽

JC Quindry ◽

MB Hudson ◽

JT Selsby

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Disease Progression ◽

Gene Mutations ◽

Dystrophin Gene ◽

Mdx Mice ◽

Dystrophin Deficiency ◽

Muscle Wasting Disease ◽

Dystrophin Protein

Background Duchenne muscular dystrophy is a muscle wasting disease caused by dystrophin gene mutations resulting in dysfunctional dystrophin protein. Autophagy, a proteolytic process, is impaired in dystrophic skeletal muscle though little is known about the effect of dystrophin deficiency on autophagy in cardiac muscle. We hypothesized that with disease progression autophagy would become increasingly dysfunctional based upon indirect autophagic markers. Methods Markers of autophagy were measured by western blot in 7-week-old and 17-month-old control (C57) and dystrophic (mdx) hearts. Results Counter to our hypothesis, markers of autophagy were similar between groups. Given these surprising results, two independent experiments were conducted using 14-month-old mdx mice or 10-month-old mdx/Utrn± mice, a more severe model of Duchenne muscular dystrophy. Data from these animals suggest increased autophagosome degradation. Conclusion Together these data suggest that autophagy is not impaired in the dystrophic myocardium as it is in dystrophic skeletal muscle and that disease progression and related injury is independent of autophagic dysfunction.

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Differential effects of dystrophin and utrophin gene transfer in immunocompetent muscular dystrophy (mdx) mice

Physiological Genomics ◽

10.1152/physiolgenomics.2000.3.3.133 ◽

2000 ◽

Vol 3 (3) ◽

pp. 133-144 ◽

Cited By ~ 37

Author(s):

SATORU EBIHARA ◽

GHIABE-HENRI GUIBINGA ◽

RENALD GILBERT ◽

JOSEPHINE NALBANTOGLU ◽

BERNARD MASSIE ◽

...

Keyword(s):

Immune System ◽

Muscular Dystrophy ◽

Gene Transfer ◽

Tibialis Anterior Muscle ◽

High Stress ◽

Dystrophin Gene ◽

Foreign Protein ◽

Mdx Mice ◽

Host Immune System ◽

Differential Effects

Ebihara, Satoru, Ghiabe-Henri Guibinga, Renald Gilbert, Josephine Nalbantoglu, Bernard Massie, George Karpati, and Basil J. Petrof. Differential effects of dystrophin and utrophin gene transfer in immunocompetent muscular dystrophy (mdx) mice. Physiol Genomics 3: 133–144, 2000.—Duchenne muscular dystrophy (DMD) is a fatal disease caused by defects in the gene encoding dystrophin. Dystrophin is a cytoskeletal protein, which together with its associated protein complex, helps to protect the sarcolemma from mechanical stresses associated with muscle contraction. Gene therapy efforts aimed at supplying a normal dystrophin gene to DMD muscles could be hampered by host immune system recognition of dystrophin as a “foreign” protein. In contrast, a closely related protein called utrophin is not foreign to DMD patients and is able to compensate for dystrophin deficiency when overexpressed throughout development in transgenic mice. However, the issue of which of the two candidate molecules is superior for DMD therapy has remained an open question. In this study, dystrophin and utrophin gene transfer effects on dystrophic muscle function were directly compared in the murine (mdx) model of DMD using E1/E3-deleted adenovirus vectors containing either a dystrophin (AdV-Dys) or a utrophin (AdV-Utr) transgene. In immunologically immature neonatal animals, AdV-Dys and AdV-Utr improved tibialis anterior muscle histopathology, force-generating capacity, and the ability to resist injury caused by high-stress contractions to an equivalent degree. By contrast, only AdV-Utr was able to achieve significant improvement in force generation and the ability to resist stress-induced injury in the soleus muscle of immunocompetent mature mdx animals. In addition, in mature mdx mice, there was significantly greater transgene persistence and reduced inflammation with utrophin compared to dystrophin gene transfer. We conclude that dystrophin and utrophin are largely equivalent in their intrinsic abilities to prevent the development of muscle necrosis and weakness when expressed in neonatal mdx animals with an immature immune system. However, because immunity against dystrophin places an important limitation on the efficacy of dystrophin gene replacement in an immunocompetent mature host, the use of utrophin as an alternative to dystrophin gene transfer in this setting appears to offer a significant therapeutic advantage.

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Mini- and full-length dystrophin gene transfer induces the recovery of nitric oxide synthase at the sarcolemma of mdx4cv skeletal muscle fibers

Gene Therapy ◽

10.1038/sj.gt.3300553 ◽

1998 ◽

Vol 5 (1) ◽

pp. 59-64 ◽

Cited By ~ 21

Author(s):

A Decrouy ◽

J-M Renaud ◽

JA Lunde ◽

G Dickson ◽

BJ Jasmin

Keyword(s):

Nitric Oxide ◽

Skeletal Muscle ◽

Nitric Oxide Synthase ◽

Gene Transfer ◽

Muscle Fibers ◽

Dystrophin Gene ◽

Full Length ◽

Skeletal Muscle Fibers ◽

Oxide Synthase

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Coxsackievirus and adenovirus receptor (CAR)-positive immature osteoblasts as targets of adenovirus-mediated gene transfer for fracture healing

Gene Therapy ◽

10.1038/sj.gt.3302060 ◽

2003 ◽

Vol 10 (18) ◽

pp. 1623-1628 ◽

Cited By ~ 16

Author(s):

T Ito ◽

K Tokunaga ◽

H Maruyama ◽

H Kawashima ◽

H Kitahara ◽

...

Keyword(s):

Gene Transfer ◽

Fracture Healing ◽

Coxsackievirus And Adenovirus Receptor ◽

Adenovirus Receptor

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Ectodomain of Coxsackievirus and Adenovirus Receptor Genetically Fused to Epidermal Growth Factor Mediates Adenovirus Targeting to Epidermal Growth Factor Receptor-Positive Cells

Journal of Virology ◽

10.1128/jvi.74.15.6875-6884.2000 ◽

2000 ◽

Vol 74 (15) ◽

pp. 6875-6884 ◽

Cited By ~ 137

Author(s):

Igor Dmitriev ◽

Elena Kashentseva ◽

Buck E. Rogers ◽

Victor Krasnykh ◽

David T. Curiel

Keyword(s):

Growth Factor ◽

Gene Transfer ◽

Epidermal Growth Factor ◽

Targeted Delivery ◽

Attachment Site ◽

Soluble Form ◽

Target Cells ◽

Epidermal Growth ◽

Coxsackievirus And Adenovirus Receptor ◽

Adenovirus Receptor

ABSTRACT Human adenovirus (Ad) is extensively used for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency.

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