Bernd Hauck

Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

Leber's congenital amaurosis (LCA) is a group of inherited blinding diseases with onset during childhood. One form of the disease, LCA2, is caused by mutations in the retinal pigment epithelium-specific 65-kDa protein gene (RPE65). We investigated the safety of subretinal delivery of a recombinant adeno-associated virus (AAV) carrying RPE65 complementary DNA (cDNA) (ClinicalTrials.gov number, NCT00516477 [ClinicalTrials.gov]). Three patients with LCA2 had an acceptable local and systemic adverse-event profile after delivery of AAV2.hRPE65v2. Each patient had a modest improvement in measures of retinal function on subjective tests of visual acuity. In one patient, an asymptomatic macular hole developed, and although the occurrence was considered to be an adverse event, the patient had some return of retinal function. Although the follow-up was very short and normal vision was not achieved, this study provides the basis for further gene therapy studies in patients with LCA.

Although most animal experiments with recombinant adeno-associated virus (AAV) vectors have been based on AAV serotype 2, recent studies showed that AAV vectors based on AAV serotype 1 performed more efficiently in muscle and other tissues. On the other hand, AAV2-based vectors can be readily purified by heparin column. To combine the advantages of both types of vectors, we developed a strategy to generate chimeric vectors by using a mixture of AAV helper plasmids encoding both serotypes in the transfection process. Because the AAV packaging machinery cannot distinguish between closely related AAV1 and AAV2 capsid proteins, each packaged virion contains capsid proteins from both serotypes. As expected, the resulting chimeric vectors could be purified by heparin column. Neutralizing antibody assays showed that the chimeric vectors can be inhibited by either AAV1 or AAV2 antiserum. In vivo, the chimeric vectors direct levels of expression similar to those of AAV1 in muscle or AAV2 in liver; that is, they combine the best transduction characteristics of both parent vectors. In summary, this study provides a straightforward method for combining various properties of different AAV serotypes into one vector. Potential limitations of the chimeric vectors are also discussed.