Detailed Protocol for the Novel and Scalable Viral Vector Upstream Process for AAV Gene Therapy Manufacturing.

Recombinant adeno-associated viral (rAAV) vector-based gene therapy has been adapted for use in more than 100 clinical trials. This is mainly because of its excellent safety profile, ability to target a wide range of tissues, stable transgene expression, and significant clinical benefit. However, the major challenge is to produce a high-titer, high-potency vector to achieve a better therapeutic effect. Even though the three plasmid-based transient transfection method is currently being used for AAV production in many clinical trials, there are complications associated with scalability and it is not cost-effective. Other methods require either large-scale production of two herpes simplex viruses, rHSV-RepCap and rHSV-GOI (gene of interest), with high titers, or a stable cell line with high titer wild-type adenovirus infection. Both of these options make the process even more complex. To address this issue, we have developed a stable cell line-based production with the use of only one rHSV-RepCap virus. Using this new methodology in small-scale production, we achieved ∼1-6 E + 04 vg/cell of AAV9 in the top producer clones. Large-scale production in 10-CS (10-Cell Stack) of one of the top producing clones resulted in ∼1-2 E + 13 vg/10-CS with 50% of full capsid ratio after purification. This method could potentially be adapted to suspension cells. The major advantage of this novel methodology is that by using the rHSV-RepCap virus, high titer AAV can be produced with any GOI containing a stable adherent or suspension producer cell line. The use of this AAV production platform could be beneficial for the treatment of many diseases.

Immediate Interpretation and Results Communication Decreases Patient Anxiety: Experience in a Private Practice Community Hospital.

OBJECTIVE. The study objectives were to evaluate the percentage of patients interested in immediate radiologic results and to measure patient anxiety and hospital loyalty with this program in a private practice community hospital setting. SUBJECTS AND METHODS. Between December 2018 and May 2019, 100 patients 18-89 years old with a primary diagnosis of abdominal pain had imaging orders for CT, ultrasound, or nuclear medicine-hepatobiliary iminodiacetic acid scans and subsequently were asked if they wanted immediate results. Those who opted for immediate results were given a two-question survey before and after results delivery that asked about their anxiety level and imaging center loyalty. Data were entered using SurveyMonkey (SVMK) and analyzed using SAS software (version 9.4, SAS Institute). RESULTS. The majority (78%) of patients wanted immediate results. There were statistically significant differences in anxiety level before and after results delivery; 37% (p < 0.001) reported decreased anxiety after receiving imaging results, whereas 57% reported no change in anxiety (p < 0.001). The decision whether to come back to this imaging center (hospital loyalty) did not change before and after test results; 85% of participants strongly agreed that they would return. CONCLUSION. It is feasible for private practice radiologists to discuss CT, ultrasound, or nuclear medicine imaging results with patients in a community hospital setting. The majority of patients preferred immediate results over traditional methods of notification. Most patients reported anxiety about their imaging results, and a statistically significant number had decreased anxiety after discussing results with radiologists.