Recent hemophilia B clinical trials using adeno-associated virus (AAV) gene delivery have demonstrated much lower FIX production in patients compared to the high levels observed in animal models and AAV capsid specific CTLs response elicited at high doses of AAV vectors. These results emphasize the necessity to explore effective approaches for enhancement of AAV transduction. Initially, we found that incubation of all AAV vectors with human serum enhanced AAV transduction. Complementary analytical experiments demonstrated that human serum albumin (HSA) directly interacted with the AAV capsid and augmented AAV transduction. The enhanced transduction was observed with clinical grade HSA. Mechanistic studies suggest that HSA increases AAV binding to target cells and that the interaction of HSA with AAV doesn’t interfere with the AAV infection pathway. Importantly, HSA incubation during vector dialysis also increased transduction. Finally, HSA enhancement of AAV transduction in a model of hemophilia B displayed greater than a 5-fold increase in vector derived circulating FIX, which improved the bleeding phenotype correction. In conclusion, incubation of HSA with AAV vectors supports a universal augmentation of AAV transduction and more importantly, this approach can be immediately transitioned to the clinic for the treatment of hemophilia and other diseases.

Patients with neutralizing antibodies (Nab) against adeno-associated virus (AAV) are usually excluded from treatment with AAV vectors. To develop a standard assay for detecting Nab inhibition activity, we systematically studied current AAV Nab assays in vitro and in vivo. Several factors were found that influence the Nab titers based on the in vitro assay, including: sera volume, AAV dose/cell, cell number and choice of transgenes. When the Nab titer assay was performed in vivo via intramuscular (IM) or systemic administration, a 4-fold increase in sensitivity for measurement of Nab titers was observed compared to an identical in vitro test. To better mimic the clinical setting, after passively transferring human Nabs into mice, blood was collected before systemic injection of AAV vector and used for Nab titer analysis in vitro or via IM injection. The results showed that AAV delivered via IM injection had a similar inhibition pattern to systemic administration. These studies indicate critical parameters necessary for optimizing Nab sensitivity and that an in vivo Nab assay is more sensitive than an in vitro assay for inclusion/exclusion criteria. The variables identified by this study may explain some of the compounding clinical data seen to date with respect to efficiency of AAV transduction in various Phase I clinical trials.

A 7-year-old male with hemoglobin SS disease and alpha thalassemia was admitted in preparation for a bone marrow transplant.Past medical history included repeated episodes of pain crisis, acute chest syndrome (ACS), silent infarcts, and a remote history of asthma, which was well controlled without medication. He presented for admission in his normal state of health and without complaints.The patient received one unit of RBCs by simple transfusion prior to placement of a temporary apheresis catheter. Surgery was uneventful (Figure S1 for chest X-ray post surgery). Postoperatively, the patient was awake with stable vitals and mild surgical pain. A few hours later, he had an episode of coughing and throat discomfort, which resulted in oxygen desaturations. Examination of the mouth and oral pharynx showed no edema or swelling. Chest X-ray at this time showed increased haziness in right lung fields (Figure S1). Presumed diagnosis was splinting due to postoperative pain leading to atelectasis.