AAV9 delivering a modified human Mullerian inhibiting substance as a gene therapy in patient-derived xenografts of ovarian cancer

Abstract

To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurrences are particularly needed. Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells. We have recently engineered peptide modifications to human MIS [albumin leader Q425R MIS (LRMIS)] that increase production and potency in vitro and in vivo. To test this novel therapeutic peptide, serous malignant ascites from highly resistant recurrent ovarian cancer patients were isolated and amplified to create low-passage primary cell lines. Purified recombinant LRMIS protein successfully inhibited the growth of cancer spheroids in vitro in a panel of primary cell lines in four of six patients tested. Adeno-associated virus (AAV) -delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression. Therefore, AAV9 was used as a single i.p. injection to deliver LRMIS to test its efficacy in inhibiting growth of palpable tumors in patient-derived ovarian cancer xenografts from ascites (PDXa). AAV9-LRMIS monotherapy resulted in elevated and sustained blood concentrations of MIS, which significantly inhibited the growth of three of five lethal chemoresistant serous adenocarcinoma PDXa models without signs of measurable or overt toxicity. Finally, we tested the frequency of MIS type II receptor expression in a tissue microarray of serous ovarian tumors by immunohistochemistry and found that 88% of patients bear tumors that express the receptor. Taken together, these preclinical data suggest that AAV9-LRMIS provides a potentially well-tolerated and effective treatment strategy poised for testing in patients with chemoresistant serous ovarian cancer.varian cancer is an enigmatic disease with 70% recurrence, and it is almost invariably fatal, even after complete response to chemotherapy and debulking i.p. surgery. Newer agents, such as angiogenesis inhibitors (1, 2) or ADP-ribose polymerase inhibitors (3), have only provided short-term improvement to survival outcomes. A dire need exists to develop novel therapeutic strategies addressing those highly chemoresistant recurrences that drive mortality. Epithelial ovarian cancers have long been known as "Mullerian" tumors, because histopathology of serous cystadenocarcinoma, endometrioid, and mucinous carcinomas recapitulates the embryonic Mullerian duct, the anlagen of the fallopian tube, uterus, cervix, and upper vagina (4). Hence, we turned to Mullerian inhibiting substance (MIS; also known as anti-Mullerian hormone), an evolutionarily conserved endogenous hormone of fetal and adult gonads (5), as a therapeutic against Mullerian-derived ovarian cancer. Recombinant human MIS (rhMIS) produced using a genomic clone (6) and purified from CHO serum-free media (7, 8) caused regression of fetal Mullerian ducts ex vivo and inhibited mouse ovarian cancer cell lines generated by Misr2-Cre directing T antigen in vitro and in vivo (9, 10). Similar effects were evident in established h...

The anti-tumor efficacy of 3C23K, a glyco-engineered humanized anti-MISRII antibody, in an ovarian cancer model is mainly mediated by engagement of immune effector cells

“…The choice of a recombinant MISRII protein was based on the observation that MISRII expression in cell lines derived from OC and OC ascites rapidly and progressively decreases after long-term culture [21], thus limiting the result reproducibility. Compared with chimeric (ch12G4), the binding capacity of h12G4 was about 20-fold reduced.…”

AMH/MIS as a contraceptive that protects the ovarian reserve during chemotherapy

“…Recombinant AAV9 (rAAV) viral particles were produced, purified, and titrated as previously described (22). Briefly, HEK293 cells were transfected with an AAV plasmid (pAAV) containing the LR-MIS construct flanked with inverted terminal repeats, and the virus was rescued with plasmids expressing AAV9 capsid proteins and adenoviral helper proteins.…”

“…To address these concerns, we created a cDNA construct (21) in which we replaced the Leader sequence with that of albumin and introduced a Q425R substitution in the C-terminal cleavage site to produce an MIS protein analog (LR-MIS, which we will refer to simply as "rhMIS"), with increased activating cleavage and secretion. The LR-MIS transgene also was used to produce an optimized adeno-associated virus serotype 9 (AAV9) gene therapy vector compatible with in vivo use, which we will refer to as "AAV9-MIS" (22).…”

Anti-Müllerian Hormone Signaling Regulates Epithelial Plasticity and Chemoresistance in Lung Cancer

“…This may be particularly relevant considering the continued clinical development of therapeutics to target TGF-β superfamily members and the association of EMT with chemoresistance (Akhurst and Hata, 2012; Fischer et al, 2015; Thiery et al, 2009; Zheng et al, 2015). Targeting AMH signaling has been proposed as a potential treatment for EOC, either through administration of exogenous AMH (Kim et al, 2014), adeno-associated virus (AAV)-delivery of AMH (Pepin et al, 2015), or use of AMHR2-targeting antibodies (Kersual et al, 2014). The present study suggests that modulating AMH homeostasis may also have effect in non-gynecological tumors.…”