TSC1 Gene Therapy AAVerts Neurological Associated Mortality
Authors: David M. Feliciano
Journal: Molecular Therapy (2019)

Tuberous sclerosis complex (TSC) is a rare genetic disorder characterized by a myriad of congenital growths called hamartomas that are found along the neurocutaneous axis. Brain lesions are correlated with severe neurological complications, which are a significant cause of morbidity and mortality. Gaining knowledge of the genetic defects responsible for TSC (loss-of-function mutations in TSC1 or TSC2 genes)1European Chromosome 16 Tuberous Sclerosis ConsortiumIdentification and characterization of the tuberous sclerosis gene on chromosome 16.Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1508) Google Scholar, 2van Slegtenhorst M. de Hoogt R. Hermans C. Nellist M. Janssen B. Verhoef S. Lindhout D. van den Ouweland A. Halley D. Young J. et al.Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.Science. 1997; 277: 805-808Crossref PubMed Scopus (1393) Google Scholar has enabled lines of research that significantly contributed to the understanding of normal and pathological brain development. Interestingly, while mutations in TSC1 or TSC2 can be inherited, the majority spontaneously arise in discrete regions through somatic cell inactivation, causing mosaic patterns of abnormal cells and focal lesions. This complexity makes therapy challenging, and, thus, complementary therapeutic approaches are needed. In Molecular Therapy - Methods & Clinical Development, Prabhakar et al.3Prabhakar S. Cheah P.S. Zhang X. Zinter M. Gianatasio M. Hudry E. Bronson R.T. Kwiatkowski D.J. Stemmer-Rachamimov A. Maguire C.A. et al.Long-term Therapeutic Efficacy of Intravenous AAV-Mediated Hamartin Replacement in Mouse Model of Tuberous Sclerosis Type 1.Mol. Ther. Methods Clin. Dev. 2019; https://doi.org/10.1016/j.omtm.2019.08.003Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar report that TSC1 gene replacement therapy in juveniles is sufficient to rescue premature death in a mouse model of TSC. TSC1 and TSC2 encode for the proteins hamartin and tuberin, respectively. Tuberin is a GTPase activating protein (GAP) that forms a complex with hamartin, which prevents the GTPase Rheb from activating mammalian target of rapamycin (mTOR).4Krueger D.A. Northrup H. International Tuberous Sclerosis Complex Consensus GroupTuberous sclerosis complex surveillance and management: recommendations of the 2012 International Tuberous Sclerosis Complex Consensus Conference.Pediatr. Neurol. 2013; 49: 255-265Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar mTOR is a protein kinase that phosphorylates proteins that regulate events ranging from anabolic protein translation to autophagy.5Saxton R.A. Sabatini D.M. mTOR Signaling in Growth, Metabolism, and Disease.Cell. 2017; 168: 960-976Abstract Full Text Full Text PDF PubMed Scopus (3161) Google Scholar Therefore, it should come as no surprise that TSC brain lesions are comprised of cytomegalic (enlarged) neurons and giant cells. Clinical trials validated the concept of mTOR inhibition to treat TSC, and this paradigm is now in common use.6Sahin M. Henske E.P. Manning B.D. Ess K.C. Bissler J.J. Klann E. Kwiatkowski D.J. Roberds S.L. Silva A.J. Hillaire-Clarke C.S. et al.Tuberous Sclerosis Complex Working Group to Update the Research PlanAdvances and future directions for tuberous sclerosis complex research: Recommendations from the 2015 strategic planning conference.Pediatr. Neurol. 2016; 60: 1-12Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar However, mTOR inhibitors are not without problems, owing to the need for continual treatment, risk of infection due to the immune suppressive effects, and moderate efficacy. These limitations fueled interest in development of a gene therapy. The first step toward this goal is the generation of a suitable animal model. To this end, Prabhakar et al.3Prabhakar S. Cheah P.S. Zhang X. Zinter M. Gianatasio M. Hudry E. Bronson R.T. Kwiatkowski D.J. Stemmer-Rachamimov A. Maguire C.A. et al.Long-term Therapeutic Efficacy of Intravenous AAV-Mediated Hamartin Replacement in Mouse Model of Tuberous Sclerosis Type 1.Mol. Ther. Methods Clin. Dev. 2019; https://doi.org/10.1016/j.omtm.2019.08.003Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar subjected neonatal mice with conditional Tsc1 alleles containing loxP sites to dual intracerebroventricular injection of adeno-associated virus vector (AAV)-encoding CRE recombinase.3Prabhakar S. Cheah P.S. Zhang X. Zinter M. Gianatasio M. Hudry E. Bronson R.T. Kwiatkowski D.J. Stemmer-Rachamimov A. Maguire C.A. et al.Long-term Therapeutic Efficacy of Intravenous AAV-Mediated Hamartin Replacement in Mouse Model of Tuberous Sclerosis Type 1.Mol. Ther. Methods Clin. Dev. 2019; https://doi.org/10.1016/j.omtm.2019.08.003Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar In this way, mosaic Tsc1 deletion creates a stochastic TSC model.7Prabhakar S. Goto J. Zhang X. Sena-Esteves M. Bronson R. Brockmann J. Gianni D. Wojtkiewicz G.R. Chen J.W. Stemmer-Rachamimov A. et al.Stochastic model of Tsc1 lesions in mouse brain.PLoS ONE. 2013; 8: e64224Crossref PubMed Scopus (14) Google Scholar Tsc1 deletion in cells surrounding the ventricles, hippocampus, and cortex, including neurons and astrocytes, reduced the median survival of mice to 38 days. Once mice with disease phenotype had been generated, effectiveness of a gene therapy could be tested. Cohorts of AAV1-Cre injected mice were also subjected to a single retro-orbital intravenous injection at 3 weeks of age with AAV8-GFP control, AAVrh8-TSC1, or an AAV9 double-stranded vector encoding TSC1. The survival of mice injected with AAV-TSC1 was remarkably improved compared to that of control mice. AAV9-TSC1 injected mice lived over 400 days and recovered motor function that was lost after Tsc1 deletion. The cause of death of AAV1-Cre injected mice that did not receive gene therapy is not precisely known, but mice exhibited macrocephaly, hunched backs associated with distress, ventriculomegaly, a domed skull, and loss of motor skills. These observations led the authors to suggest that hydrocephalus might be responsible. As with TSC patients, Tsc1 deletion in subventricular zone (SVZ) neural stem cells generated migratory heterotopias and subependymal nodules (SENs).7Prabhakar S. Goto J. Zhang X. Sena-Esteves M. Bronson R. Brockmann J. Gianni D. Wojtkiewicz G.R. Chen J.W. Stemmer-Rachamimov A. et al.Stochastic model of Tsc1 lesions in mouse brain.PLoS ONE. 2013; 8: e64224Crossref PubMed Scopus (14) Google Scholar, 8Feliciano D.M. Quon J.L. Su T. Taylor M.M. Bordey A. Postnatal neurogenesis generates heterotopias, olfactory micronodules and cortical infiltration following single-cell Tsc1 deletion.Hum. Mol. Genet. 2012; 21: 799-810Crossref PubMed Scopus (52) Google Scholar, 9Zhou J. Shrikhande G. Xu J. McKay R.M. Burns D.K. Johnson J.E. Parada L.F. Tsc1 mutant neural stem/progenitor cells exhibit migration deficits and give rise to subependymal lesions in the lateral ventricle.Genes Dev. 2011; 25: 1595-1600Crossref PubMed Scopus (86) Google Scholar In TSC patients, SENs can become subependymal giant cell astrocytomas (SEGAs), which are type I neoplastic lesions. SEGAs are found along the ventricular walls of the brain and can prevent cerebrospinal fluid flow, causing hydrocephalus, migraines, and seizures. Consistent with the notion of SENs leading to hydrocephalus, the SVZ of Tsc1 deleted mice was enlarged due to a greater number of proliferative cells. Importantly, changes to the ventricular walls and ventricles were rescued by AAV9-TSC1. The SVZ contains neural stem cells and transits amplifying cells, microglia, ependyma, neuroblasts, and astrocytes. Therefore, identifying the cells that are proliferating and determining whether this is a cell autonomous effect is important. Tubers are another type of lesion commonly found within the cerebral cortex of TSC patients,6Sahin M. Henske E.P. Manning B.D. Ess K.C. Bissler J.J. Klann E. Kwiatkowski D.J. Roberds S.L. Silva A.J. Hillaire-Clarke C.S. et al.Tuberous Sclerosis Complex Working Group to Update the Research PlanAdvances and future directions for tuberous sclerosis complex research: Recommendations from the 2015 strategic planning conference.Pediatr. Neurol. 2016; 60: 1-12Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar comprising disorganized areas of the brain containing abnormal cells. Tuber number, size, and clinical imaging features often underlie severe and pharmacologically refractory forms of seizures that are diagnosed within the first years of life.4Krueger D.A. Northrup H. International Tuberous Sclerosis Complex Consensus GroupTuberous sclerosis complex surveillance and management: recommendations of the 2012 International Tuberous Sclerosis Complex Consensus Conference.Pediatr. Neurol. 2013; 49: 255-265Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar Loss of TSC genes within neurons commonly leads to seizures in mice. Although overt signs of seizures were not seen in these mice, it is known from experience with patients that infantile spasms and absence seizures can be difficult to detect (absence seizures cause children to blank out or stare into space for a few seconds). AAV1-Cre targets cortical and hippocampal neurons and astrocytes that could cause seizures when Tsc1 is deleted. Alternatively, enlarged neurons or astrocytes could potentially lead to megalencephaly and hydrocephalus. Thus, determining the exact cause of death will be important to provide mechanistic insight into how AAV9-TSC1 rescue is exerted. TSC1 and TSC2 appear to have conserved functions in numerous species, as exemplified by the conserved phenotypes shared between TSC conditional mice and many of the manifestations seen in patients. The fact that human TSC1 rescues mouse Tsc1 phenotypes is profound and supports the continued use of this model. However, clinical gene therapy might prove to be more nuanced. For example, many mutations in TSC1 do not simply result in loss of hamartin expression. Mutations that result in dominant negative function could alter tuberin or Rheb, and it is unclear whether AAV-delivered hamartin could compensate. Thus, successful clinical trials would need to carefully choose patient cohorts with TSC1 mutations. Nonetheless, the findings reported by Prabhakar et al.3Prabhakar S. Cheah P.S. Zhang X. Zinter M. Gianatasio M. Hudry E. Bronson R.T. Kwiatkowski D.J. Stemmer-Rachamimov A. Maguire C.A. et al.Long-term Therapeutic Efficacy of Intravenous AAV-Mediated Hamartin Replacement in Mouse Model of Tuberous Sclerosis Type 1.Mol. Ther. Methods Clin. Dev. 2019; https://doi.org/10.1016/j.omtm.2019.08.003Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar represent a significant advance in the fight against TSC and reinforce the need for complementary therapeutic strategies that replace TSC1 and TSC2 functions. D.M.F. is supported by grants from the Whitehall Foundation (2015-08-05) and the NIH (1R15NS096562).