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Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy. Here we describe a novel trans-splicing strategy, which targets highly abundant pre-mRNAs, to produce therapeutic proteins in vivo. First, we used a pre-trans-splicing molecule (PTM) that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-mRNA and production of hapoA-I protein. The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice. Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo.

Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology for the repair of defective pre-messenger RNA (pre-mRNA) molecules. It is especially useful in the treatment of genetic disorders involving large genes. Although viral vectors have been used for achieving long-lasting expression of trans-splicing molecules, the immunogenicity and suboptimal safety profiles associated with viral-based components could limit the widespread application of SMaRT in the repair of genetic defects. Here, we tested whether the non-viral Sleeping Beauty (SB) transposon system could mediate stable delivery of trans-splicing molecules designed to correct the genetic defect responsible for severe combined immune deficiency (SCID). This immunological disorder is caused by a point mutation within the 12.4 kilobase (kb) gene encoding the DNA protein kinase catalytic subunit (DNA-PKcs) and is associated with aberrant DNA repair, defective T- and B-cell production, and hypersensitivity to radiation-induced injury. Using a novel SB-based trans-splicing vector, we demonstrate stable mRNA correction, proper DNA-PKcs protein production, and conference of a radiation-resistant phenotype in a T-cell thymoma cell line and SCID multipotent adult progenitor cells (MAPCs). These results suggest that SB-based trans-splicing vectors should prove useful in facilitating the correction of endogenous mutated mRNA transcripts, including the DNA-PKcs defect present in SCID cells.

The recent recommendation of the Board of Directors of the American Society of Gene and Cell Therapy that the National Institutes of Health (NIH) Recombinant DNA Advisory Committee (RAC) end its review of clinical protocols in human gene therapy and human gene marking is a welcome and necessary step in the continuing evolution of the oversight of clinical gene therapy.1 In the late 1980s and early 1990s, the RAC and the Division of Cell and Gene Therapy of the Center for Biologics Evaluation and Research at the US Food and Drug Administration (FDA) met often in informal sessions following RAC meetings to discuss and review various aspects of clinical trials in gene marking and therapy. Indeed, RAC review was a productive part of the process when these early gene transfer protocols were being considered. This pooling of expertise was a completely new approach to therapeutics that greatly aided both groups. However, owing in large part to the emergence of a strong and capable full-time group devoted to cell and gene therapy within the FDA, RAC review is no longer necessary.

Low levels of high density lipoprotein (HDL) represent an important predictor of cardiovascular disease risk1,2. An increase of 1mg/dl in HDL correlates with a risk reduction of cardiovascular disease of 2|[ndash]|3%3. We have adapted spliceosome mediated RNA trans-splicing technology (SMaRT|[trade]|) to trans-splice the sequences of wild type human apolipoprotein A-I (apoA-I) into the highly abundant albumin pre-mRNA in hepatocytes with the objective of transiently increasing blood levels of HDL. ApoA-I is the major protein component of HDL. We targeted a pre-trans-splicing molecule (PTM) to bind to specific sequences in the first intron of mouse albumin pre-mRNA. The bound PTM will then trans-splice, inserting sequences for human ApoA-I into intron 1 of albumin pre-mRNA. With trans-splicing, apoA-I would be made in hepatocytes, the same cells that naturally synthesize apoA-I.

Cardiovascular disease (CVD) is the most common cause of death in Western societies, and its worldwide prevalence is increasing. A very strong predictor of CVD risk is the plasma concentration of high-density lipoprotein (HDL) or of its major protein component, ApoA-1. HDL and ApoA-1 levels exhibit an inverse relationship with the development of atherosclerosis and coronary heart disease1,2,3 and exogenous ApoA1 and HDL protect against atherosclerosis and plaque development 4,5,6,7. The present study was undertaken to determine if RNA trans-splicing could be used to increase blood levels of human ApoA-1 (hApoA1). To this end we targeted a pre-trans-splicing molecule (PTM) encoding human ApoA1 to the highly abundant albumin pre-messenger RNA (pre-mRNA). Trans-splicing between the PTM and the albumin pre-mRNA results in the production of human ApoA1 protein with two residual albumin amino acids. This fusion protein was expressed, processed and secreted as well as authentic hApoA1 and indeed the fusion protein had full activity in ATP-binding cassette transporter protein (ABC1)-mediated transfer of cellular cholesterol efflux assays.

Adenosine phosphorylase (adenosine: orthophosphate ribosyltransferase) activity is low in mammalian cells, but is abundant in certain prokaryotes. It is an appropriate target enzyme for the development of diagnostic and chemotherapeutic agents. Adenosine phosphorylase from Bacillus subtilis and mycoplasmas that commonly infect cell cultures converted the non-toxic deoxyadenosine analog, 6-methylpurine deoxyriboside (6MPDR) into the potent anti-metabolites 6-methylpurine and 6-methylpurine riboside. Consequently 6MPDR selectively killed mammalian cell cultures infected with mycoplasmas. 6MPDR was cytotoxic to 87 of 90 mycoplasma-infected cultures (96.6%). No toxicity was observed in nine different types of mycoplasma-free cell cultures. Use of a 3T6 mouse embryo fibroblast indicator cell culture improved standardization. Cytotoxicity was apparent 3–4 days after inoculation of 10 μM of 6MPDR to 3T6 cultures infected with the following mycoplasmas: M. hyorhinis, M. orale, M. arginini, M. salivarium, A, laidlawii, M. hominis, M. fermentons, M. sp . 70–159 and M. buccale . Cytotoxicity was produced in 28/28 3T6 indicator cultures inoculated with mycoplasma-infected cell cultures in the presence of 10 μM 6MPDR. The analog is apparently non-toxic to the mycoplasmas.

A simple and rapid method of species identification of mycoplasmas by immunobinding assay is described. Small amounts of antigen of supernatant from cell cultures, broth cultures or clinical specimens were spotted onto nitrocellulose paper. This was followed by application of specific anti-mycoplasma antisera. After incubation, an enzyme-conjugated antiserum against the first antiserum was applied. A positive reaction was indicated by the development of intense blue color reaction when substrate was added. This method identified mycoplasma species with monoclonal and polyclonal antibodies. It detected 9.3 X 10(3) - 7.5 X 10(4) CFU/ml of organisms depending on mycoplasma species. For identification of mycoplasma, ureaplasma, acholeplasma and spiroplasma species, this assay is useful and rapid compared with other serological methods. In limited studies, the method correlated with microbiological assay of clinical specimens for Mycoplasma pneumoniae.