Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.

Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.

Measuring the outcome of health resort programs.

OBJECTIVE: To evaluate the metric properties and practicability of valid, internationally available outcome instruments in the special setting of health resort programs. METHODS: A cohort study in a convenience sample of patients with low back pain, upper back pain, conditions of the lower extremities, and conditions of the upper extremities was conducted. Their functioning and health were assessed before and after a health resort program by the disease-specific North American Spine Society (NASS) instruments Lumbar NASS and Cervical NASS; WOMAC Osteoarthritis Index; Disabilities of Arm, Shoulder and Hand Questionnaire; and the general instrument, Medical Outcome Study Short Form-36 (SF-36). RESULTS: Completeness on the scale level ranged between 1% and 10%. Criterion validity of condition-specific instruments was confirmed by stronger associations of the pain and function scales to the Physical Health component of the SF-36 (r = -0.59 to -0.79, p < 0.001 for all scales) than to the Mental Health component (r = -0.11, NS, to r = -0.42, p < 0.001). Reliability (Cronbach's alpha coefficient) was higher than 0.8 for all scales of condition-specific instruments and for 6 of 8 SF-36 scales. Floor and ceiling effects ranged between 0% and 7%. The condition-specific instruments demonstrated a good responsiveness with an effect size ranging between 0.28 and 0.55 and with a standardized response mean between 0.32 and 0.94. The responsiveness of most SF-36 scales was similar, but the Physical Function scale showed a lower responsiveness than the condition-specific scales. CONCLUSION: The evaluated instruments can be recommended for use in clinical trials that assess the outcome of health resort programs.

MIA (melanoma inhibitory activity) promoter mediated tissue-specific suicide gene therapy of malignant melanoma.

Suicide gene therapy of malignant melanoma essentially requires efficient gene transfer and highly selective therapeutic gene expression. To achieve this, recombinant adeno-associated virus (rAAV) particles were constructed containing the tissue-specific promoter of the human melanoma inhibitory activity (hMIA) gene combined with four copies of the enhancer element of the murine tyrosinase gene. Three melanoma and one cervix carcinoma cell line were infected with rAAV particles carrying a reporter gene under control of the enhancer/hMIA promoter in order to determine transcriptional activity and specificity of this system. Viral particles containing the enhancer/hMIA promoter mediated reporter gene activity only in melanoma cells, whereas infection with a cytomegalovirus (CMV)-based promoter construct induced unspecific gene expression. Correspondingly, transient transduction with viral particles bearing the HSVtk gene under the control of the enhancer/MIA promoter elements followed by treatment with ganciclovir (GCV) resulted in growth inhibition only in melanoma cells, whereas the CMV promoter-based construct induced unspecific cytotoxicity. In vivo experiments in nude mice demonstrated that tumors originating from human melanoma cells disappeared after stable, but not transient transduction with vectors bearing the HSVtk gene under the control of the enhancer/hMIA promoter in response to GCV application. In face of higher transduction efficiency, these rAAV particles might therefore be a useful tool for suicide gene therapy of malignant melanoma.

Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6.

We present a simple and safe strategy for producing high-titer adeno-associated virus (AAV) vectors derived from six different AAV serotypes (AAV-1 to AAV-6). The method, referred to as "HOT," is helper virus free, optically controllable, and based on transfection of only two plasmids, i.e., an AAV vector construct and one of six novel AAV helper plasmids. The latter were engineered to carry AAV serotype rep and cap genes together with adenoviral helper functions, as well as unique fluorescent protein expression cassettes, allowing confirmation of successful transfection and identification of the transfected plasmid. Cross-packaging of vector DNA derived from AAV-2, -3, or -6 was up to 10-fold more efficient using our novel plasmids, compared to a conservative adenovirus-dependent method. We also identified a variety of useful antibodies, allowing detection of Rep or VP proteins, or assembled capsids, of all six AAV serotypes. Finally, we describe unique cell tropisms and kinetics of transgene expression for AAV serotype vectors in primary or transformed cells from four different species. In sum, the HOT strategy and the antibodies presented here, together with the reported findings, should facilitate and support the further development of AAV serotype vectors as powerful new tools for human gene therapy.