In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement.

Targeted genetic correction of mutations in cells is a potential strategy for treating human conditions that involve nonsense, missense, and transcriptional splice junction mutations. One method of targeted gene repair, single-stranded short-fragment homologous replacement (ssSFHR), has been successful in repairing the common deltaF508 3-bp microdeletion at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in 1% of airway epithelial cells in culture. This study investigates in vitro and in vivo application of a double-stranded method variant of SFHR gene repair to the mdx mouse model of Duchenne muscular dystrophy (DMD). A 603-bp wild-type PCR product was used to repair the exon 23 C-to-T mdx nonsense transition at the Xp21.1 dys locus in cultured myoblasts and in tibialis anterior (TA) from male mdx mice. Multiple transfection and variation of lipofection reagent both improved in vitro SFHR efficiency, with successful conversion of mdx to wild-type nucleotide at the dys locus achieved in 15 to 20% of cultured loci and in 0.0005 to 0.1% of TA. The genetic correction of mdx myoblasts was shown to persist for up to 28 days in culture and for at least 3 weeks in TA. While a high frequency of in vitro gene repair was observed, the lipofection used here appeared to have adverse effects on subsequent cell viability and corrected cells did not express dystrophin transcript. With further improvements to in vitro and in vivo gene repair efficiencies, SFHR may find some application in DMD and other genetic neuromuscular disorders in humans.

Fasciola: kinetics and quality of humoral responses to fatty acid binding protein and cathepsin l following delivery as DNA vaccines in mice.

The humoral response to DNA vaccination of mice with two important Fasciola antigens has been investigated. Both F. gigantica fatty acid binding protein (FABP) and F. hepatica cathepsin L5 (FhCatL5) were shown to be expressed in COS 7 cells and induced a humoral response when delivered as secretory constructs in mice. FABP induced an IgGl dominant response, with significant IgE, IgG2a, and IgG2b responses also present, indicating a mixed Th1/Th2 response. The total Ig response peaked at 1:24,500 and antibody titers were sustained for at least 32 weeks. In contrast, the delivery of FABP as a nonsecreted construct did not result in the induction of a measurable humoral response. FhCatL5 was delivered as a secretory construct, with secretion mediated by the native F. hepatica signal sequence, which was shown to operate in COS 7 cells. The humoral response peaked at 1:2000 at week 8 and was sustained for at least 20 weeks. Antibody isotype analysis demonstrated a Th2-like response, which was qualitatively different from that obtained for FABP with an IgE dominant response, and lower titers to IgG1 and IgG3. The results demonstrate that Fasciola antigens can be delivered as DNA vaccines, but that the quality of the response varies between antigens and is influenced by the method of vaccine delivery.

Targeted gene correction in the mdx mouse using short DNA fragments: towards application with bone marrow-derived cells for autologous remodeling of dystrophic muscle.

In muscle, mutant genes can be targeted and corrected directly by intramuscular (i.m.) injection of corrective DNA, or by ex vivo delivery of DNA to myogenic cells, followed by cell transplantation. Short fragment homologous replacement (SFHR) has been used to repair the exon 23 nonsense transition at the Xp21.1 dys locus in cultured cells and also, directly in tibialis anterior from male mdx mice. Whilst mdx dys locus correction can be achieved in up to 20% of cells in culture, much lower efficiency is evident by i.m. injection. The major consideration for application of targeted gene correction to muscle is delivery throughout relevant tissues. Systemically injected bone marrow (BM)-derived cells from wt C57BL/10 ScSn mice are known to remodel mdx muscle when injected into the systemic route. Provided that non muscle-derived cell types most capable of muscle remodeling activity can be more specifically identified, isolated and expanded, cell therapy seems presently the most favorable vehicle by which to deliver gene correction throughout muscle tissues. Using wt bone marrow as a model, this study investigates systemic application of bone marrow-derived cells as potential vehicles to deliver corrected (ie wt) dys locus to dystrophic muscle. Intravenous (i.v.) and intraperitoneal (i.p.) injections of wt BM were given to lethally and sub-lethally irradiated mdx mice. Despite both i.v. and surviving i.p. groups containing wt dys loci in 100% and less than 1% of peripheral blood nuclei, respectively, both groups displayed equivalent levels of wt dys transcript in muscle RNA. These results suggest that the muscle remodeling activity observed in systemically injected BM cells is not likely to be found in the hemopoietic fraction.

Humoral responses in mice following vaccination with DNA encoding glutathione S-transferase of Fasciola hepatica: effects of mode of vaccination and the cellular compartment of antigen expression.

The humoral responses in mice following vaccination with DNA constructs encoding Fasciola hepatica glutathione S-transferase (GST) have been evaluated. GST47 cDNA was subcloned into two DNA vaccine vectors, VR1012 and VR1020, which direct expression to the cytoplasmic and extracellular compartments, respectively. Expression was confirmed by transfection into COS 7 cells. Groups of mice were vaccinated with these constructs, by either intramuscular injection with the VR1012-or VR1020-based constructs, or intradermal vaccination (with a gene gun) with the VR1020-based construct. Vaccination with the construct designed for secretion resulted in an increased humoral response compared to vaccination with the nonsecretory construct. The level of the total humoral response after vaccination with the secretion construct was not dependent on the route of vaccination. However, the isotype profile of the response differed between the groups; intramuscular vaccination with the construct directing cytoplasmic expression yielded an immuoglobulin (Ig)G2a dominant (Th1-type) response, intradermal vaccination with the secretory construct a IgG1/IgE dominant (Th2-type) response, and intramuscular vaccination with the secretory construct a mixed isotype response. These results demonstrate that the immunogenicity of a DNA vaccine based on Fasciola GST, as well as the isotype of the response against GST, is determined by the mode of vaccine administration.