Partial Rescue of Growth Failure in Growth Hormone (GH)-Deficient Mice by a Single Injection of a Double-Stranded Adeno-Associated Viral Vector Expressing the GH Gene Driven by a Muscle-Specific Regulatory Cassette

Abstract

Growth hormone (GH) deficiency (GHD) causes somatic growth impairment. GH has a very short half-life and therefore it has to be administered by daily subcutaneous injections. Adeno-associated viral (AAV) vectors have been used to deliver genes to animals, and recently developed double stranded (ds)–AAV vectors provide widespread and stable transgene expression. In the present study we tested whether an intramuscular injection of ds-AAV vector expressing GH under the control of an M-creatine kinase regulatory cassette would assure sufficient systemic GH delivery in conjunction with muscle-specific expression. Virus-injected GHD mice showed a significant (p<0.05) increase in body length and body weight, without reaching full normalization, and significant (p<0.05) reduction in absolute and relative visceral fat. Quantitative RT-PCR showed preferential GH expression in skeletal muscles that was confirmed by qualitative fluorescence analysis in mice injected with a similar virus expressing GFP. The present study shows that systemic GH delivery to GHD animals is possible via a single intramuscular injection of dsAAV carrying a muscle-specific GH-expressing regulatory cassette.

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

“…To further enhance muscle-specific expression, we used an AAV9-Cas9 vector (CK8e-Cas9-shortPolyA), which contains a muscle-specific CK regulatory cassette, referred to as the CK8e promoter, which is highly specific for expression in the muscle and the heart (Fig. 2D) (33). Together, this 436-bp muscle-specific cassette and the 4101-bp Cas9 complementary DNA (cDNA) are within the packaging limit of AAV9 (32, 34).…”

“…pGL3-CK8e plasmid has been previously described (33). A full description of this small regulatory cassette will be reported elsewhere.…”

CRISPR-induced deletion with SaCas9 restores dystrophin expression in dystrophic models in vitro and in vivo

“…Even if this off-target site is located within a non-coding segment of the IL-17A gene, strategies to limit possible adverse effects would have to be considered. A possible strategy would be to use a skeletal muscle and cardiac tissue specific promoter, such as the CK8e promoter developed by Hauschka's laboratory 21,26,44 , which restricts the expression of the nuclease to tissues where editing is required to improve the DMD patient phenotype.…”

Different ex Vivo and Direct in Vivo DNA Administration Strategies for Growth Hormone Gene Therapy in Dwarf Animals

“…More recently, in 2008, a new generation of double-stranded adeno-associated viral vectors (dsAAV) encoding mGH cDNA driven by a universal promoter (CMV) were used by a group coordinated by Johns Hopkins University School of Medicine to prepare viral particles that were injected into GHRHKO mice, a model of isolated GH deficiency due to generalized ablation (knock-out, KO) of the GHRH gene 17,18 . These genetically modified dsAAV can infect dividing and non-dividing cells in vitro and in vivo with a long-term transgenic expression (up to 1 year) and elicit a lower toxicity and cellular immune response, therefore being generally considered to be safer than adenoviral vectors.…”

“…Such systems could be based on the use of inducible promoters that can be regulated at will or of tissue-specific promoters, providing good systemic delivery together with limited local expression. In a subsequent study, the same group utilized a dsAAV expressing mGH cDNA under the control of a muscle creatine kinase regulatory cassette in order to ensure adequate systemic delivery in conjunction with muscle-specific expression 18 . A low-dose (0.5 x 10 11 pfu) and a high-dose (1 x 10 11 pfu) of virus were injected into the right quadriceps muscle of GHRHKO mice at the age of day 10.…”