Local administration of AAV-DJ pseudoserotype expressing COX2 provided early onset of transgene expression and promoted bone fracture healing in mice.

We have previously obtained compelling proof-of-principle evidence for COX2 gene therapy for fracture repair using integrating retroviral vectors. For this therapy to be suitable for patient uses, a suitable vector with high safety profile must be used. Accordingly, this study sought to evaluate the feasibility of AAV as the vector for this COX2 gene therapy, because AAV raises less safety issues than the retroviral vectors used previously. However, an appropriate AAV serotype is required to provide early increase in and adequate level of COX2 expression that is needed for fracture repair. Herein, we reported that AAV-DJ, an artificial AAV pseudoserotype, is highly effective in delivering COX2 gene to fracture sites in a mouse femoral fracture model. Compared with AAV-2, the use of AAV-DJ led to ~5-fold increase in infectivity in mesenchymal stem cells (MSCs) and provided an earlier and significantly higher level of transgene expression at the fracture site. Injection of this vector at a dose of 7.5 × 10(11) genomic copies led to high COX2 level at the fracture site on day 3 after injections and significantly promoted fracture union at 21 days, as analyzed by radiography and µ-CT. The therapeutic effect appears to involve enhanced osteoblastic differentiation of MSCs and remodeling of callus tissues to laminar bone. This interpretation is supported by the enhanced expression of several key genes participating in the fracture repair process. In conclusion, AAV-DJ is a promising serotype for the AAV-based COX2 gene therapy of fracture repair in humans.

Genetic variation in bone-regenerative capacity among inbred strains of mice.

Genetic variation in bone-regenerative capacity has not been studied in any animal model system. We developed a "drill-hole" model in the tail vertebra of inbred strains of mice that allows us to reproducibly introduce an injury with a defined boundary and quantify the rate of bone healing using the combination of high-resolution Faxitron X-ray imaging and the ChemiImager 4000 Low Light Imaging System. Using this model, we demonstrate that bone-regenerative capacity is a genetically controlled trait with an estimated heritability of 72%, and that it differs significantly among inbred strains of mice. Of the 12 inbred strains tested, Sencar/PtJ was identified as the most suitable model for the study of hard-tissue regeneration. This strain regained 73% of bone loss 30 days after injury, in contrast to the slow healer, CBA/J, which recovered only 25% of the bone loss during the same period. Bone-regenerative capacity was not correlated with soft-tissue-regenerative capacity, suggesting that different sets of genes may regulate soft- and hard-tissue regeneration. It was, however, significantly correlated with total bone mineral density (R = 0.49, p < 0.01), indicating that high bone density is associated not only with prevention of bone fracture, but also with promotion of bone regeneration.

Developmental expression of the murine Prl-1 protein tyrosine phosphatase gene.

The expression of the murine Prl-1 protein tyrosine phosphatase gene was examined in normal embryos from E10.5 through E18.5. Prl-1 mRNA was detected in the brain, neural tube, and dorsal root ganglia, and in several non-neuronal tissues, including the skeletal system. Heart and skeletal muscle were consistently negative. At E13.5, Prl-1 was expressed in the condensing prechondrogenic cells of the vertebrae, whereas at E18.5, Prl-1 mRNA was localized to the hypertrophic chondrocytes. The dynamic expression of Prl-1 during cartilage differentiation may suggest a functional role in skeletal development.

In situ hybridization analysis of immunoglobulin heavy chain variable gene expression with family specific oligonucleotide probes.

We have developed an improved in situ hybridization (ISH) technique for the analysis of human immunoglobulin heavy chain variable (V(H)) gene family expression in suspensions of human B lymphocytes. Oligonucleotide probes specific for framework region (FR) consensus germline sequences for each of the seven human V(H) gene families were designed and hybridization conditions were developed to accommodate the greatest degree of V(H) gene variation, maximize the sensitivity of transcript detection, and assure the specificity of the technique. The hybridization parameters were rigorously characterized by Southern hybridization to a panel of 30 V(H) cDNA clones and by ISH to 17 B cell lines expressing characterized V(H) genes. Results obtained with ISH using V(H) gene family and isotype-specific gene probes correlated well with histochemical measures of Ig gene product expression. Profiles of cellular V(H) gene expression were generated for mitogen stimulated peripheral blood B lymphocytes from six normal subjects. When compared with estimates of frequency of V(H) genes in the human germline, the results were consistent with a random pattern of V(H) family utilization.

Transactivation of Hox gene expression in a VP16-dependent binary transgenic mouse system.

Mice with aberrant expression of Hox genes have provided valuable insight into the role of Hox class transcription factors in patterning the developing skeleton and the nervous system. However, a recurrent problem is the lethality of mice expressing a Hox-transgene. To circumvent premature death frequently associated with transgenes that interfere with development, we have established a binary transgenic mouse system. Transactivator mice harbor the VP16 gene regulated by a promoter of interest while transresponder mice contain the VP16-responsive immediate early (IE) promoter linked to the gene to be expressed [G.W. Byrne, F.H. Ruddle, Multiplex gene regulation: a two-tiered approach to transgene regulation in transgenic mice, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5473-5477]. Here, we report the generation of transresponder mouse strains that harbor murine homeobox genes linked to the IE promoter. We provide evidence that these transgenes are transcriptionally activated in progeny that inherit both a transactivator and transresponder transgene. By microdissection of mouse embryos and reverse transcription polymerase chain reaction (RT-PCR) analysis, we demonstrate that the expression of the Hox-transgenes is restricted to those regions of the mouse embryos where VP16 is present. The ability to activate stable Hox-transgenes in a reproducible fashion now permits a detailed in vivo dissection of the molecular mechanisms that lead to developmental abnormalities caused by deregulated Hox-gene expression.

Immunoglobulin gene expression in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by inflammation of synovium, in which immunoglobulin-secreting plasma cells are generally present. The forces driving immunoglobulin expression in RA synovium are unknown. Sequences of VH and VK transcripts from an RA synovial cDNA library demonstrate patterns of somatic mutation typical of an antigen-driven response. Moreover, 5% of the kappa repertoire appears to derive from the same B cell progenitor, suggesting an oligoclonal response. Immunoglobulin expression in this synovium thus appears to result from antigen stimulation. In addition, this patient's synovium is enriched for unusually long VK-JK joins (CDR3s), suggesting abnormal selection or regulation of the B cell response in RA.