We tested a new in vivo hematopoietic stem cell (HSC) transduction/selection approach in rhesus macaques using HSCtropic, integrating, helper-dependent adenovirus vectors (HDAd5/35++) designed for the expression of human g-globin in red blood cells (RBCs) to treat hemoglobinopathies. We show that HDAd5/35++ vectors preferentially transduce HSCs in vivo after intravenous injection into granulocyte colony-stimulating factor (G-CSF)/AMD3100-mobilized animals and that transduced cells return to the bone marrow and spleen. The approach was well tolerated, and the activation of proinflammatory cytokines that are usually associated with intravenous adenovirus vector injection was successfully blunted by pre-treatment with dexamethasone in combination with interleukin (IL)-1 and IL-6 receptor blockers. Using our MGMT P140K -based in vivo selection approach, g-globin + RBCs increased in all animals with levels up to 90%. After selection, the percentage of g-globin + RBCs declined, most likely due to an immune response against human transgene products. Our biodistribution data indicate that g-globin + RBCs in the periphery were mostly derived from mobilized HSCs that homed to the spleen. Integration site analysis revealed a polyclonal pattern and no genotoxicity related to transgene integrations. This is the first proof-of-concept study in nonhuman primates to show that in vivo HSC gene therapy could be feasible in humans without the need for high-dose chemotherapy conditioning and HSC transplantation.

We developed a new in vivo hematopoietic stem cell (HSC) gene therapy approach that involves only intravenous injections and does not require myeloablation/conditioning and HSC transplantation. In this approach, HSCs are mobilized from the bone marrow into the peripheral blood stream and transduced with intravenously injected helper-dependent adenovirus (HDAd) vectors. A fraction of transduced HSCs returns to the bone marrow and persists there long-term. Here, we report desmoglein 2 (DSG2) as a new receptor that can be employed for in in vivo HSC transduction. We developed HDAd5/3+ vectors that use DSG2 as a high-affinity attachment receptor and studied in vivo HSC transduction and safety after intravenous injection of an HDAd5/3+GFP vector in G-CSF/AMD3100(Plerixafor)-mobilized rhesus macaques. Unlike previously used CD46-targeting HDAd5/35++ vectors, HDAd5/3+ virions were not sequestered by rhesus erythrocytes and therefore mediated ~10-fold higher GFP marking rates in primitive HSCs (CD34+/CD45RA-/CD90+ cells) in the bone marrow at day 7 after vector injection. To further increase the return of in vivo transduced, mobilized HSCs to the bone marrow, we transiently expressed CXC motif chemokine receptor 4 (cxcr4) in mobilized HSCs from the HDAd5/3+ vector. In vivo transduction with a HDAd5/3+GFP/cxcr4 vector at a low dose of 0.4x1012vp/kg resulted in up to 7% of GFP-positive CD34+/CD45RA-/CD90+ cells in the bone marrow. This transduction rate is a solid basis for in vivo base or prime editing in combination with natural or drug-induced expansion of edited HSCs. Furthermore, our study provides new insights into HSC biology and trafficking after mobilization in non-human primates.

While SARS-CoV2 vaccines have shown an unprecedented success, the ongoing emergence of new variants and necessity to adjust vaccines justify the development of alternative prophylaxis and therapy approaches. Hematopoietic stem cell (HSC) gene therapy using a secreted CoV2 decoy receptor protein (sACE2-Ig) would involve a one-time intervention resulting in long-term protection against airway infection, viremia, and extrapulmonary symptoms. We recently developed a technically simple and portable in vivo hematopoietic HSC transduction approach that involves HSC mobilization from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating, helper-dependent adenovirus (HDAd5/35 ++ ) vector system. Considering the abundance of erythrocytes, in this study, we directed sACE2-Ig expression to erythroid cells using strong β-globin transcriptional regulatory elements. We performed in vivo HSC transduction of CD46-transgenic mice with an HDAd-sACE2-Ig vector. Serum sACE2-Ig levels reached 500–1,300 ng/mL after in vivo selection. At 22 weeks, we used genetically modified HSCs from these mice to substitute the hematopoietic system in human ACE2-transgenic mice, thus creating a model that is susceptible to SARS-CoV2 infection. Upon challenge with a lethal dose of CoV2 (WA-1), sACE2-Ig expressed from erythroid cells of test mice diminishes infection sequelae. Treated mice lost significantly less weight, had less viremia, and displayed reduced cytokine production and lung pathology. The second objective of this study was to assess the safety of in vivo HSC transduction and long-term sACE2-Ig expression in a rhesus macaque. With appropriate cytokine prophylaxis, intravenous injection of HDAd-sACE2-Ig into the mobilized animal was well tolerated. In vivo transduced HSCs preferentially localized to and survived in the spleen. sACE2-Ig expressed from erythroid cells did not affect erythropoiesis and the function of erythrocytes. While these pilot studies are promising, the antiviral efficacy of the approach has to be improved, for example, by using of decoy receptors with enhanced neutralizing capacity and/or expression of multiple antiviral effector proteins.

Our goal is to overcome treatment resistance in ovarian cancer patients which occurs in most cases after an initial positive response to chemotherapy. A central resistance mechanism is the maintenance of desmoglein-2 (DSG2) positive tight junctions between malignant cells that prevents drug penetration into the tumor. We have generated JO4, a recombinant protein that binds to DSG2 resulting in the transient opening of junctions in epithelial tumors. Here we present studies toward the clinical translation of c-JO4 in combination with PEGylated liposomal doxorubicin/Doxil for ovarian cancer therapy. A manufacturing process for cGMP compliant production of JO4 was developed resulting in c-JO4. GLP toxicology studies using material from this process in DSG2 transgenic mice and cynomolgus macaques showed no treatment-related toxicities after intravenous injection at doses reaching 24 mg/kg. Multiple cycles of intravenous c-JO4 plus Doxil (four cycles, 4 weeks apart, simulating the treatment regimen in the clinical trial) elicited antibodies against c-JO4 that increased with each cycle and were accompanied by elevation of pro-inflammatory cytokines IL-6 and TNFα. Pretreatment with steroids and cyclophosphamide reduced anti-c-JO4 antibody response and blunted cytokine release. Our data indicate acceptable safety of our new treatment approach if immune reactions are monitored and counteracted with appropriate immune suppression.

Our goal is to overcome treatment resistance in ovarian cancer patients, which occurs in most cases after an initial positive response to chemotherapy. A central resistance mechanism is the maintenance of desmoglein-2 (DSG2)-positive tight junctions between malignant cells, which prevents drug penetration into the tumor. We generated JO4, a recombinant protein that binds to DSG2, resulting in the transient opening of junctions in epithelial tumors. Here, we present studies on the clinical translation of JO4 in combination with PEGylated liposomal doxorubicin/Doxil® for ovarian cancer therapy. A manufacturing process for cGMP-compliant production of JO4 was developed. GLP toxicology studies using material from this process in DSG2 transgenic mice and cynomolgus macaques showed no treatment-related toxicities after intravenous injection at doses reaching 24 mg/kg. Multiple cycles of intravenous JO4 plus Doxil® (4 cycles, 4 weeks apart, simulating the treatment regimen in the clinical trial) elicited antibodies against JO4 that increased with each cycle and were accompanied by elevation of pro-inflammatory cytokines IL6 and TNF. Pretreatment with steroids and cyclophosphamide reduced the anti-JO4 antibody response and blunted cytokine release. Our data indicate acceptable safety of our new treatment approach if immune reactions are monitored and counteracted with appropriate immune suppression.