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Cheah et al., Sci. Adv. 2021; 7 : eabb1703 8 January 2021

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D I S E A S E S A N D D I S O R D E R S

Gene therapy for tuberous sclerosis complex type 2 in a mouse model by delivery of AAV9 encoding a condensed form of tuberinPike-See Cheah1,2*, Shilpa Prabhakar1*, David Yellen1, Roberta L. Beauchamp3, Xuan Zhang1, Shingo Kasamatsu4,5, Roderick T. Bronson6, Elizabeth A. Thiele7,8, David J. Kwiatkowski9, Anat Stemmer-Rachamimov10, Bence György11,12,13, King-Hwa Ling14,15, Masao Kaneki4,5, Bakhos A. Tannous1, Vijaya Ramesh3, Casey A. Maguire1, Xandra O. Breakefield1†

Tuberous sclerosis complex (TSC) results from loss of a tumor suppressor gene - TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins formed a complex to inhibit mTORC1-mediated cell growth and prolifer-ation. Loss of either protein leads to overgrowth lesions in many vital organs. Gene therapy was evaluated in a mouse model of TSC2 using an adeno-associated virus (AAV) vector carrying the complementary for a “condensed” form of human tuberin (cTuberin). Functionality of cTuberin was verified in culture. A mouse model of TSC2 was generated by AAV-Cre recombinase disruption of Tsc2-floxed alleles at birth, leading to a shortened lifespan (mean 58 days) and brain pathology consistent with TSC. When these mice were injected intravenously on day 21 with AAV9-cTuberin, the mean survival was extended to 462 days with reduction in brain pathology. This demon-strates the potential of treating life-threatening TSC2 lesions with a single intravenous injection of AAV9-cTuberin.

INTRODUCTIONTuberous sclerosis complex (TSC) is a hereditary disease affecting multiple organs with an incidence of about 1 of 5500 (1, 2), resulting from mutations in either TSC1 encoding hamartin or TSC2 encoding tuberin. Hamartin and tuberin normally act as a complex to inhibit mTORC1 (mammalian/mechanistic target of rapamycin complex 1) through guanosine triphosphatase (GTPase) activating effects on Ras homolog enriched in brain (Rheb) (3). When a mutation in the corresponding normal TSC1 or TSC2 allele occurs somatically in susceptible cells, they enlarge and proliferate causing abnormal de-velopment and tissue lesions. These secondary mutations can occur prenatally or after birth in different cell types, and the timing and frequency of these hits affect the severity of the disease in a stochastic manner. Neurodevelopmental manifestations are responsible for the greatest morbidity, including severe, refractory epilepsy and hydrocephalus, as well as autism (40%), cognitive impairment

(50%), and mental health issues (70%) (4–6). In addition, renal angiomyolipomas forming later in life can cause life-threatening hemorrhage and/or renal failure, and pulmonary lymphangioleio-myomatosis can severely compromise respiratory function. Current treatments include surgical resection and/or treatment with rapa-mycin analogs (rapalogs). Although often well tolerated, rapalogs cause immune suppression (7) and potentially compromise early brain development (8), and lifelong therapy is often required. Therefore, there is a clear need to identify other therapeutic ap-proaches for TSC.

Adeno-associated virus (AAV) vectors have been used widely in clinical trials for many hereditary diseases with little-to-no toxicity, long-term action in nondividing cells, and improvement in symp-toms (9–11). Benefit can be seen after a single injection and some serotypes, e.g., AAV9, AAVrh8, and AAVrh10, can efficiently enter the brain, as well as peripheral organs after intravenous (IV) injec-tion (12, 13). The insert capacity of AAV vectors is about 4.7 kb (including promoter, transgene, polyadenylation (poly A) se-quence, and other regulatory elements), and the complementary DNA (cDNA) for tuberin (5.4 kb) cannot be accommodated. We generated a cDNA encoding a shorter form of tuberin, termed cTuberin. We tested its lack of toxicity and ability to bind to hamartin and Rheb1, as well as to suppress phosphoS6 kinase activity in cul-tured cells. In a stochastic mouse model of TSC2 [based on a TSC1 model; (14)], AAV vector encoding Cre recombinase was intro-duced by intracerebroventricular (ICV) injection into homozygous Tsc2-floxed mice (15) at postnatal day 0 (P0) typically leading to death at about P58 with enlarged ventricles. Near-normal life span and reduction of brain pathology were achieved in most of these animals by a single IV injection of an AAV9 vector encoding cTuberin under a strong, constitutive promoter. These studies demonstrate the ability of cTuberin to suppress overgrowth of tuberin-null cells, including neural cells and, presumably, other cells in the body, and, hence, support the preclinical efficacy of AAV-cTuberin for TSC2 lesions.

1Molecular Neurogenetics Unit, Department of Neurology and Center for Molecu-lar Imaging Research, Department of Radiology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, Boston, MA, USA. 2Depart-ment of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia. 3Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA. 4Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA. 5Shriners Hospitals for Children, Boston, MA, USA. 6Rodent Histopathology Core Facility, Harvard Medical School, Boston, MA, USA. 7Herscot Center for Tuberous Sclerosis Complex, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. 8The Pediatric Epilepsy Program, Department of Neurology, Massachusetts General Hospital, Boston, MA, USA. 9Brigham and Women’s Hospital, Harvard Med-ical School, Boston, MA, USA. 10Department of Pathology, Massachusetts General Hospital, Boston, MA, USA. 11Department of Neurobiology and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA. 12Institute of Mo-lecular and Clinical Ophthalmology, Basel, Switzerland. 13Department of Oph-thalmology, University of Basel, Basel, Switzerland. 14Department of Genetics, Harvard Medical School, Boston, MA, USA. 15Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia.*These authors contributed equally to this work as co-first authors.†Corresponding author. Email: [email protected]

Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

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RESULTScTuberin constructWhereas hamartin is encoded in a cDNA of 3.5 kb, which fits into an AAV vector (16), the cDNA for tuberin (5.4 kb) is too large. To generate a potentially functional form of tuberin encoded in a shorter cDNA, we retained the N-terminal domain that binds to hamartin and the C-terminal domain containing GAP (GTPase- activating protein) activity that inhibits Rheb, with N-terminal region and phosphorylation of the C-terminal region of tuberin also thought to regulate formation of the complex with hamartin Fig. 1A (3, 17–20). The potential for cTuberin to retain some functional activity was supported by findings of Momose et al. (21) that genomic overexpression of the C-terminal region of rat tuberin (amino acids 1425 to 1755) can suppress renal tumors in the Tsc2 Eker rat model. We felt it was also important to retain the hamartin-binding domain at the N terminus, as hamartin and tuberin function together as a complex with Tre2-Bub2-Cdc16 (TBC) 1 domain family, member 7 (TBC1D7) to accelerate guanosine triphosphate (GTP) to guanosine diphosphate conversion of Rheb-GTP (3, 22). In addition, this re-quirement for complex formation for activity might act to limit poten-tial negative effects of high levels of transgenic cTuberin expression. cTuberin was thus designed to retain key elements of function, in-cluding 450 amino acids from the N-terminal region and 292 amino acids from the C-terminal region, joined by a flexible serine-glycine linker of 16 amino acids (fig. S1). This cDNA, with a Kozak sequence, and a C-terminal c-Myc tag was inserted into an AAV2 backbone under a chicken -actin (CBA) promoter (23), with a WPRE (wood-chuck hepatitis virus posttranscriptional regulatory element) and poly A signals (Fig. 1B).

cTuberin is expressed in cultured cells with no apparent toxicityHuman embryonic kidney (HEK) 293T cells were transfected with plasmids for empty AAV (AAV1-null that contains all the elements

except the cTuberin cDNA), AAV-CBA–green fluorescent protein (GFP), or AAV-CBA-cTuberin-Myc to assess the expression level of cTuberin. In addition to endogenously expressed tuberin (200 kDa), cTuberin expression at the appropriate molecular weight (MW) of 85 kDa was detected on Western blots using anti-tuberin and anti-Myc antibodies (Fig. 2A; representative blot, n = 3) Immunocytochem-istry of 293T cells transfected with different plasmids demonstrated stronger tuberin immunoreactivity in those transfected with AAV-CBA-cTuberin-Myc compared to other groups that expressed only endogenous tuberin (Fig. 2B; representative micrographs, n = 3). We determined transfection efficiency of these cells in two ways—microscopically and by flow cytometry. As it is challenging to dif-ferentiate expression of endogenous tuberin from cTuberin, image analysis was carried out microscopically for each well (approximately 2000 cells per well; n = 3) for 4′,6-diamidino-2-phenylindole (DAPI)– positive and c-Myc–positive cells, and we determined that 43 ± 2% of cells were transfected with the AAV-CBA-cTuberin-Myc (Fig. 2B). Cytotoxicity assays were also performed following transfection of HEK293T cells with AAV-null, AAV-CBA-GFP, or AAV-CBA-cTuberin-Myc plasmids to evaluate potential toxicity of cTuberin. The lactate dehydrogenase (LDH) assay (Dojindo Molecular Technologies Inc., Rockville, MD, USA) revealed no cytotoxicity in cTuberin-transfected cells, as compared to controls (Fig. 2C; n = 3). As a second way to evaluate the extent of transfection of these 293T cells with AAV-CBA-cTuberin- Myc plasmid DNA (n = 3), we sorted the c-Myc–positive cells using flow cytometry, after staining the cells with unlabeled c-Myc primary antibody followed by Alexa Fluor 647–conjugated secondary antibody. Compared to the background in nontransfected cells (4 ± 1%), we detected a marked increase of c-Myc–positive cells (50 ± 1% or 46% minus the background, similar to the 43% determined by cell counting) after transfection with the AAV-CBA-cTuberin-Myc plasmid (P



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with Flag-hamartin and HA-S6K plasmids. Hamartin and full-length tuberin coexpression inhibited phosphorylation of S6K T389, as expected, and similarly, coexpression of hamartin and cTuberin also decreased pS6K T389 levels (Fig. 4A), supporting the ability of cTuberin to bind to hamartin and efficaciously inhibit TORC1 activity. Level of pS6K T389 inhibition was quantified rela-tive to HA-S6K using Fiji/ImageJ. Flag-hamartin cotransfected with AAV-GFP served as a control (normalized to 1.0), and cotransfec-tion with full-length tuberin and cTub-Myc revealed a significant inhibition of S6K T389 phosphorylation by 69 and 56%, respectively (*P



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Different cohorts of mice were subjected to body weight mea-surement and motor function assessment starting at P21/22 for naïve, noninjected animals, AAV1-Cre ICV injected (1 × 1010 vg/kg) at P1 only or followed with AAV9-cTuberin injected (8 × 1012 vg/kg) IV at P21. Body weights of these mice from age 21 to 50 days did not differ according to treatment (Fig. 5B). Movement was assessed using an automated rotarod apparatus with accelerating rotary velocity (4 going to 64 rpm over 2 min) to assess motor skills of the mice as time of latency to fall. A significant increase in latency

was observed for the AAV1-Cre + AAV9-cTuberin as compared to the AAV1-Cre–injected mice and naive mice (Fig. 5C). During animal handling, two mice of six Tsc2-floxed AAV-Cre–injected mice (day 41) and two mice of seven Tsc2-floxed AAV-Cre–injected + AAV-cTuberin– injected mice (one each on days 47 and 50) mani-fested straub (vertical tail), humped back, and/or motor seizures, which did not, however, compromise their consequent rotarod per-formances (fig. S2).

Two other approaches were less effective at extending survival of AAV1-Cre ICV–injected Tsc2-floxed mice. In one, using a similar time scheme (fig. S3), Tsc2-floxed pups were injected with 1 × 1014 vg/kg AAV1-Cre ICV at P3 and then 3 × 1012 vg/kg of AAV1-cTuberin (in contrast to AAV9 serotype) IV at P21, with the higher amount of AAV1-Cre (without cTuberin) leading to death with a mean of 36 days and survival only being extended by AAV1-cTuberin to a mean of 54 days. This probably reflects the fact that AAV1 is less efficient at crossing the blood-brain barrier (BBB) than AAV9. In another experiment, the Tsc2-floxed pups were injected ICV with AAV1-Cre (1 × 1012 vg/kg) at P0, followed by ICV injection (in con-trast to systemic injection) of 4.5 × 1013 vg/kg of AAV9-cTuberin at P3. This approach led to median survival of 50 days in Tsc2-floxed mice without cTuberin injection, while those injected with AAV9- cTuberin had extended median survival only up to 95 days (fig. S4). This experiment raises the possibility that other lesions in the body (in addition to the brain) resulting from ICV injection of AAV1-Cre were associated with death and were not sufficiently alleviated by ICV injection of the cTuberin vector and/or that the high dose AAV-cTuberin injected ICV into P3 pups had some toxicity (26).

In naïve (normal) Tsc2-floxed mice, the ventricle is lined by a single layer of ependymal cells (Fig. 6A). Neuropathological examina-tion at P42 revealed that ICV injection of AAV1-Cre in Tsc2-floxed mice at P0 led to multiple layers of ependymal and subependymal cells lining the lateral ventricle (indicating increased proliferation of these cells) (Fig. 6B, asterisk), which sometimes appeared as nodules along the ventricular lining (Fig. 6C). When these AAV1-Cre–injected mice were treated with AAV9-cTuberin (IV injected at P21), there was apparent regression of ependymal/subependymal overgrowths

Fig. 3. cTuberin-Myc binds hamartin and Rheb comparably to full-length tuberin. Representative blot (n = 3 experiments) after cotransfection of the Myc-tagged cTuberin (AAV-CBA-cTub-Myc) or full-length tuberin (Myc-FL-tuberin) along with FLAG-tagged hamartin and HA-tagged GST-Rheb1. Coimmunoprecipitation (co-IP) using anti-Myc antibody demonstrated that cTuberin-Myc interacts with both Flag-hamartin and HA-Rheb1 similar to Myc-FL-tuberin. Conversely, negative con-trol Myc-GSK-3 showed no interaction with FLAG-hamartin or HA-GST-Rheb1.

Fig. 4. cTuberin-c-Myc inhibits mTORC1 signaling comparably to full-length tuberin. (A) Full-length Flag-tagged tuberin (Flag-tuberin), Myc-tagged cTuberin (AAV-cTub-Myc), or AAV-GFP plasmids were cotransfected into HEK293T cells along with full-length Flag-tagged hamartin (Flag-hamartin) and HA-tagged p70S6K (HA-p70S6K), which is phosphorylated at T389 by mTORC1 (latter used as a reporter for mTORC1 activation). Representative blot (n = 3 experiments) demonstrated similar inhibition levels of phosphorylated p70S6K (pS6K T389) with either full-length tuberin or cTub-Myc cotransfected with full-length hamartin. (B) Quantitation of decrease in S6K T389 phosphorylation was performed relative to HA-S6K using Fiji/ImageJ. Flag-hamartin cotransfected with AAV-GFP served as a control (normalized to 1.0), and cotransfec-tion with full-length tuberin or cTub-Myc revealed inhibition of 69 or 56%, respectively, representing the results from three experiments. *P < 0.05.


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(Fig. 6D). We also stained these mouse brain sections (P42) for Ki67 as an indication of cell proliferation. As expected, there was little-to-no proliferation of ependymal/subependymal cells lining the ventricles in the naïve brain (Fig. 7A). In contrast, after AAV1-Cre injection at P0, there was marked proliferation of these cells, includ-ing apparent migration of dividing cells into the brain parenchyma (Fig. 7B), also seen after subsequent IV injection with AAV9-null vector (Fig. 7C). In contrast, IV injection of the AAV9-cTuberin vector decreased proliferation and inward migration of Ki67+ ependymal/subependymal cells (Fig. 7D).

The brain sections (P42) were also immunostained for phospho-rylated ribosomal protein S6 (pS6). We observed low pS6 expression in the whole brain sections of the noninjected (naïve) mouse brain (Fig. 8A, top). In contrast, in AAV1-Cre ICV–injected Tsc2-floxed mice, pS6 expression was intense in many brain cells [Fig. 8, A (middle) and Bi], with the pS6-positive cells being significantly larger in size (Fig. 8Bii) and with a higher pS6 immunofluorescence signal (Fig. 8Biii). When the AAV1-Cre–injected mice were subjected to IV injection of the AAV9-cTuberin vector at P21, the pS6 immuno-reactive cells were significantly decreased in average size by 23%

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another three Tsc2-floxed animals subjected to no injections were used as controls. For quantitative polymerase chain reaction (qPCR) analysis of AAV genomes (probes and primer specific), 50 ng of DNA was used as a template, and primers and probes were designed to amplify the cTuberin in the infected animal (fig. S5). cTuberin DNA was not detected in the noninjected control group (Fig. 8D). Cycle threshold (Ct) values for the Tsc2-floxed animals injected with AAV1-Cre and AAV9-cTuberin vectors were readily detectable with approximately 30.8 ± 2.6 and 17.2 ± 0.2 cycles for brain and liver tissue, respectively (Fig. 8Di). The large difference between the AAV genomes in brain compared to liver is likely due to both the high tropism of systemically injected AAV for the liver and the relatively low dose of vector injected (9 × 1011 vg/kg). To detect cTuberin transgene expression, total RNA was extracted from the brains and livers of another set of animals, including noninjected controls; Tsc2- floxed animals injected with AAV1-Cre only, and Tsc2-floxed ani-mals injected with AAV1-Cre and AAV9-cTuberin vectors (n = 3 for all groups), with the dosage of AAV1-Cre ICV injected at P1 (1 × 1010 vg/kg) or combined with AAV9-cTuberin injected IV at P21 (1.8 × 1012 vg/kg). Quantitative reverse transcription PCR (RT-qPCR)

analysis indicated that cTuberin mRNA was undetectable in the non-injected control group and those injected with AAV1-Cre only. In contrast, in both brains and livers, we detected cTuberin mRNA in mice injected with AAV9-cTuberin at levels of Ct 36.8 ± 3 and 34.8 ± 0.5 cycles, respectively (Fig. 8Dii). We did not detect cTuberin cDNA when reverse transcriptase was omitted from the RT reac-tion, indicating that we were detecting bona fide cTuberin mRNA and not sample contamination with AAV-cTuberin genomes.

DISCUSSIONThis is the first description of an alternative mode of therapy for TSC type 2 (TSC2) involving gene replacement using an AAV vec-tor encoding a condensed form of tuberin, termed cTuberin. We developed a stochastic mouse model for central nervous system (CNS) lesions in TSC2 in which homozygous Tsc2-floxed mice (15, 27) are injected ICV in the newborn period (P0 to P3) with an AAV1 vector expressing Cre recombinase, as described for our stochastic TSC1 model (14). AAV1-Cre injection in the Tsc2-floxed model resulted in death at about 58 days. Death appeared to be due primarily to

Fig. 6. Hematoxylin and eosin histochemical staining of mouse brain with and without AAV9-cTuberin vector injection following AAV1-Cre injection. Tsc2-floxed mouse pups were either not injected (naïve) or injected ICV in both ventricles (1 × 1012 vg/kg) with an AAV1-Cre vector at P0. At 21 days, some mice were injected IV with AAV9-cTuberin (9 × 1011 vg/kg) or noninjected. At 42 days, all mice were euthanized. (A) Naïve, noninjected brain (black arrowhead indicating the choroid plexus). (B and C) Tsc2-floxed mice with AAV1-Cre at P0 and no further injection showed (B) proliferation of ependymal/subependymal cells (asterisk) and (C) subependymal nodules. (D) Little-to-no subependymal overgrowth was detected in mice receiving both the P0 AAV1-Cre ICV injection and P21 IV AAV9-cTuberin injection. Representa-tive images are shown. Magnification bar, 100 m. CC, corpus callosum; LV, lateral ventricle.


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hydrocephalus caused by ependymal/subependymal overgrowths blocking cerebrospinal fluid flow, with whole-body pathology reveal-ing no overt lesions except in the CNS. Although signs of seizures were noted in a few mice during motor performance assessment, these animals recovered normal activity. Experiments showed that IV injection of AAV9-cTuberin vector into this stochastic Tsc2-floxed mouse model on day 21 extended life span in most mice (9 of 12) to at least 450 days. Histochemical/immunohistochemical analysis of the brains supported a resulting reduction in size of ependymal/subependymal lesions, decreased proliferation of cells in the sub-ependymal zone, and reduced phosphorylation of S6 kinase driven by mTOR activity. This study offers a potential single treatment para-digm for improving the outcome of patients with TSC2.

Limitations to this stochastic Tsc2 mouse model include the fact that floxed alleles (before Cre exposure) are normal in function during prenatal development and that Cre recombinase usually knocks out both alleles in a cell at once, which is different from the case in TSC2 patients, most of whom are heterozygous for one mutant and one normal allele in most-to-all cells in their body. TSC2 heterozygosity itself may compromise some cell functions and contribute to aspects of the disease phenotype (1, 28, 29). Further, the model used here is

CNS oriented, with most pathology in the brain; whereas in TSC patients, a number of organs in addition to the brain are affected. In addition, this Tsc2 mouse model does not show all the brain abnor-malities observed in human TSC2, many of which form prenatally, such as cortical tubers, disorganized cortical lamination, dysplastic neurons, and giant cells (30). Strengths of this model are that there is loss of tuberin expression in a number of different cell types in the brain with variation for animal to animal, as occurs in patients with TSC. This is in contrast to commonly used models where Tsc2-floxed mice are mated to mice expressing Cre recombinase under a cell- specific promoter, e.g., the synapsin promoter, in which case most and only neurons lose expression at embryonic day 12.5 (31).

The central portion of tuberin that was removed to fit coding sequences into the AAV vector contains a number of phosphorylation sites that are involved in regulating mTOR activity under some circum-stances, with three of these sites bearing missense mutations associated with TSC2, suggesting that they may contribute to the disease pheno-type or create truncated, nonfunctional proteins (6). By comparison, there is an ortholog of human tuberin in Schizosaccharomyces pombe that lacks about 500 amino acids in the equivalent central region of human tuberin, suggesting that these sites are dispensable to some

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Fig. 7. Ki67 immunostaining of Tsc2-floxed mice brains. Tsc2-floxed mouse pups were either not injected (naïve) or injected ICV in both ventricles (1 × 1012 vg/kg) with an AAV1-Cre vector at P0. At 21 days, some mice were injected IV with AAV9-cTuberin (9 × 1011 vg/kg), AAV9-null (1 × 1013 vg/kg) or noninjected. At 42 days, all mice were euthanized. (A) Naïve, noninjected brain reveals little-to-no staining in the ependymal/subependymal layers. (B) Tsc2-floxed mice injected with AAV1-Cre vector only showed abnormal mitotic activity and apparent migration of cells (yellow arrows) away from the ventricular zone, as well as multiple ependymal/subependymal layers (green arrowheads) as compared to the naïve group. (C) Tsc2-floxed mice injected with AAV1-Cre vector followed by AAV9-null vector showed abnormal mitotic activity of the cells and thickening of the subventricular zone. (D) The Tsc2-floxed mice injected with AAV1-Cre and then rescued with the AAV9-cTuberin vector showed a trend toward normalization of the ependymal/subependymal layer. The corresponding brain sections were counterstained with DAPI. The yellow asterisk denotes autofluores-cence in the choroid plexus. Representative images are shown. Magnification bar, 100 m.


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functions (32). Further, some of the key Akt phosphorylation sites in mammalian tuberin are not essential in Drosophila (33), and phosphorylation sites for Akt, ribosomal protein S6 kinase, and AMP-activated protein kinase (AMPK) in the central region of human tuberin are not present in Schizosaccharomyces or Dictyostelium (34), suggesting that these sites may not be critical for function. Given the critical role of phosphorylation sites in tuberin in growth factor and cytokine signaling in mammalian cells, one would anticipate that cTuberin in TSC2-null cells would lack some of these regulatory con-trols. However, in the Eker rat model of TSC2, which is prone to renal carcinomas, the C-terminal region alone (amino acids 1425 to 1755) of rat tuberin suppresses tumor formation in a dose-dependent manner (35). Fortunately, in TSC2 patients, only a very small fraction of cells in the body suffer loss of tuberin, and most damage is done by the en-largement and proliferation of these deficient cells. Thus, if overgrowths can be suppressed by cTuberin, then that would bring therapeutic benefit for many of the symptoms of the disease, although the cells would not be fully “normalized.” So far, in cultured cells, cTuberin has been shown to bind to hamartin, and overexpression of cTuberin was not found to be toxic. cTuberin inhibited mTORC1 signaling in

these cells to the same extent as tuberin, supporting the use of cTuberin as an effective replacement for tuberin for some cell properties.

Subependymal nodules (SENs) occur in 10 to 15% of children with TSC, usually appearing after birth and being more severe in TSC2 than TSC1 (36–38). SENs can enlarge into subependymal giant cell astrocytomas (SEGAs) during the first decade of life causing obstruction of cerebrospinal fluid flow, potentially leading to life- threatening hydrocephalus, as well as endocrinopathy and visual impairment (36, 37, 39, 40). Under optimal care, infants and children with TSC are monitored for subependymal lesions by magnetic res-onance imaging (MRI) every 6 to 12 months. The two current standards of care are neurosurgical removal of SEGAs through craniotomy, which can be associated with significant morbidity (37), or treatment with rapalogs, which inhibit mTOR activity. Rapalogs have proven effective in reducing lesion size, but they require continuous treatment and have limited access to the brain after peripheral administration. Potential problems with this class of drugs include a compromise of immune function (41), interference with white matter integrity (42), and possible interference with brain development in early childhood (43). In several studies, the mTOR

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Fig. 8. pS6 immunostaining of Tsc2-floxed mice brains. Tsc2-floxed mouse pups were either not injected (naïve) or injected ICV (1 × 1012 vg/kg) with an AAV1-Cre vector at P0. At P21, some mice were injected IV with AAV9-cTuberin (9 × 1011 vg/kg) or noninjected. All were euthanized at P42. (A) Whole mouse brain sections from naïve, AAV1-Cre, and AAV1-Cre+ AAV9-cTuberin injected mice stained for pS6 and DAPI. Representative whole brain sections (scale bar, 1 mm; eight-bit–thresholded in-verted images) indicated absence of pS6 puncta in naïve group. In other groups, pS6 puncta appeared as darkened spots within the cerebral cortex and caudate putamen; high magnification inset images (scale bar, 100 m; 12-bit–thresholded inverted images). (B) pS6 analysis included puncta density (i), size (ii), and intensity (iii). *P < 0.05; n = 3. a.u., arbitrary units. (C) Compared to naïve pups, immunoblotting demonstrated AAV1-Cre–mediated decrease of tuberin (54%) and increase in pS6 (76%) in Tsc2-floxed mice injected with AAV1-Cre, relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with naïve brain as control (normalized to 1.0; *P < 0.05; n = 3). (Di) Ct values for biodistribution of AAV vector genomes in the brain and liver measured by qPCR. (Dii) Ct value of GAPDH and cTuberin cDNAs in brains of naïve animals in-jected with AAV1-Cre only or with AAV1-Cre and AAV9-cTuberin. n.d., not determined.


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pathway has been found to be critical to neurodevelopment, includ-ing neuronal growth, axonal guidance, synapse formation, and my-elination (44–46). Inhibition of mTOR by rapalogs may contribute to the observed memory dysfunction following prenatal/postnatal drug treatment in Tsc mouse models (47) and the behavioral abnor-malities in wild-type mice treated prenatally with rapamycin (48). Some physicians do not recommend the use of these drugs in children or pregnant women as “long-term effects on growth and develop-ment in pediatric patients are not fully known” (43). Although in at least one study, rapalog treatment was reported to have no signifi-cant effect on neurocognitive function or behavior in children with TSC (49).

Our premise is that current therapies for children with TSC may have associated morbidity resulting in the potential for decreased mental functions. Another therapeutic approach would be intravascular administration of an AAV vector that can cross the BBB encoding a replacement “gene” for the mutant TSC1 or TSC2 alleles. Since SENs are slow growing, there would be time to monitor their size by MRI over several months and leave open the opportunity to administer standard-of-care treatment, as needed. It is hoped that gene re-placement therapy might reduce use of more problematic standard-of-care procedures in young children and provide long-lasting benefit with a single administration. Certain serotypes of AAV, such as AAV9, are able to penetrate the BBB as well as deliver to peripheral tissues (13). Thus, with IV delivery, “extra copies” of the replace-ment gene would be provided to multiple tissues, including brain, kidney, liver, and lungs, which might reduce the likelihood that so-matic mutations in TSC genes later in life would lead to disruptive hamartomas.

Advantages of AAV gene therapy are the potential for a single vector injection yielding long-term transgene expression in non-dividing cells. It is assumed that once a tuberin analog is delivered to cells in TSC2 lesions, they would shrink and stop dividing and, hence, retain transgene expression. Gene therapy may be a viable option for infants/children with TSC to reduce potential compro-mise of brain functions caused by congenital lesions and secondary sequelae of these lesions. AAV9 vectors have been used in young mice with spinal muscular atrophy (SMA) for gene replacement of the survival motor neuron (SMN) protein using both IV (50) and intrathecal (51) gene delivery. An AAV9-SMN drug, Zolgensma (Novartis), is now U.S. Food and Drug Administration–approved for IV treatment of babies/children with SMA. Two critical aspects of successful gene therapy with AAV vectors are as follows: (i) a known target, in the case of TSC2 loss of function of tuberin; and (ii) no toxicity resulting from overexpression of the replacement protein, since levels of expression cannot at present be regulated. There is a predicted reduced chance of toxicity of cTuberin as it should only be active in a 1:1 complex with hamartin, and hamartin levels are normal in TSC2 null cells (52), with cTuberin not bound to hamartin presumably being degraded. So far, no toxic effects of cTuberin expression have been observed in cells in culture or in mice. Clinical trials should be facilitated by the ability to image reduced lesion size within months by MRI due to shrinking of cell volume and inhibition of cell proliferation, as was found in the rapalog trial for renal angiomyolipomas (53). Typically, AAV vectors are just administered once due to previous exposure to the AAV virus in life eliciting an immune response to the capsid and reduc-ing secondary transduction (54). If replacement is insufficient to reduce symptoms or new TSC2 null lesions arise later in life after

AAV gene replacement, it would still be possible to treat patients with rapalogs or possibly exoAAV (55). These studies support the potential of AAV gene therapy for TSC2, which might be especially useful in infants and children where drug inhibition of the mTOR pathway may interfere with early brain development.

MATERIALS AND METHODSAAV vector design and packagingThe AAV vector plasmid, AAV-CBA-Cre-BGHpA, was derived as described in Prabhakar et al. (16). These AAV vectors carry AAV2 inverted terminal repeat elements, and gene expression is controlled by a hybrid promoter (CBA) composed of the cytomegalovirus (CMV) immediate/early gene enhancer fused to the -actin pro-moter (23). To increase the efficiency of cTuberin translation (for future use in human gene therapy approach), cDNA encoding cTuberin was human codon-optimized before gene synthesis by GenScript Biotech (Piscataway, NJ, USA). AAV vector plasmid, AAV-CBA-cTuberin-c-Myc, was derived from the plasmid pAAV-CBA-W (56). This vector contains the CBA promoter driving cTuberin, followed by a WPRE and both SV40 and bovine growth hormone (BGH) polyadenylation (poly A) signal sequences. Our cTuberin construct contains the following: ACC (Kozak sequence) :: amino acids 1 to 450 of human tuberin::gly/ser linker :: amino acids 1515 to 1807 of human tuberin :: c-Myc tag = 2307 bp encod-ing an 85-kD protein (fig. S1). The pAAV-CBA-W, which contains the CBA promoter, WPRE, and poly A sequences, but no transgene, served as AAV-null in our studies.

AAV1 and AAV9 serotype vectors were produced by transient cotransfection of HEK293T cells by calcium phosphate precipita-tion method of vector plasmids (e.g., AAV-CBA-cTuberin-Myc), adenoviral helper plasmid pAdF6, and a plasmid encoding AAV9 (pAR9) or AAV1 (pXR1) rep and capsid genes, as previously de-scribed (57). All AAV vectors carried the identity of all PCR-amplified sequences as confirmed by sequencing. Briefly, AAV vectors were purified by iodixanol density gradient centrifugation. The virus- containing fractions were concentrated using Amicon Ultra 100-kDa molecular weight cut-offs (MWCO) centrifugal devices (EMD Millipore, Billerica, MA, USA), and the titer vector genomes (vg) per milliliter was determined by quantitative real-time PCR amplification with primers and TaqMan probe specific for the BGH poly A signal.

Cell cultureHEK293T cells [American Type Culture Collection (ATCC)] and COS-7 cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Hampton, NH, USA) supplemented with 10% fetal bovine serum (FBS; Gemini Bio Products, West Sacramento, CA, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific). The cell cultures were periodically screened to ensure they are free from mycoplasma contamination using the PCR Mycoplasma Detection Kit (ABM, G238, Richmond, BC, Canada).

Detection of cell cytotoxicity using LDH release assayHEK293T cells were seeded in 96-well plates (10,000 cells per well) and, after 24 hours, transfected with various plasmid DNAs (AAV-null, AAV-GFP, and AAV-cTuberin) at 250 ng/10,000 cells using Lipo-fectamine 2000, according to the manufacturer’s instructions (Life Technologies, Carlsbad, CA, USA) in Opti-MEM (Life Technologies).


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Six hours later, transfection media was removed and replaced with DMEM (10% FBS and 1% Penicillin-Streptomycin solution), and cells were allowed to grow for 72 hours. One group of cells was treated with potent proteasome inhibitor Bortezomib (VELCADE; Mil-lennium Pharmaceuticals Inc., Cambridge, MA, USA) (58) at 250 nM for 72 hours, as a positive control for toxicity. Cellular toxicity caused by plasmid DNA transfection was assessed by quantification of extracellular LDH activity using LDH assay kit-WST (Dojindo Molecular Technologies Inc.), following the manufacturer’s instruc-tions. Briefly, the supernatant for each transfected or treated sample was collected and incubated with substrate for 30 min at 37°C. Fol-lowing incubation, stop solution was added, and absorbance was measured at 490 nm.

Western blotsBriefly, cultured cells were harvested in lysis buffer [50 mM Hepes (pH 8.0), 150 mM NaCl, 2 mM EDTA, 2.5% sodium dodecyl sulfate, 2% CHAPS, 2.5 mM sucrose, 10% glycerol, 10 mM sodium fluoride, 2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride (Sigma- Aldrich, St. Louis, MO, USA), 10 mM sodium pyrophosphate, and protease inhibitor cocktail (P8340, Sigma-Aldrich)]. After sonica-tion and incubation at 8°C for 10 min, the samples were centrifuged at 14,000g for 30 min at 8°C. Equal amounts of protein, determined by a detergent-compatible protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), were boiled for 5 min in Laemmli sample buffer (Bio-Rad), separated by SDS–polyacrylamide gel electrophoresis (PAGE), and transferred onto nitrocellulose membranes (Bio-Rad). Equal protein loading was confirmed by Ponceau S staining. The membranes were blocked in 2% blocking reagent (GE Healthcare, Pittsburgh, PA, USA) for 1 hour at room temperature (RT) and in-cubated with primary antibodies overnight at 4°C. Anti-tuberin/TSC2 (#3612), anti–phospho-S6 (#2211), anti-S6 (#2212), anti-Myc (clone 9B11, #2276) (Cell Signaling Technology, Danvers, MA, USA), anti–-actin (#A5441), anti-FLAG (clone M2, #F1804) (Sigma- Aldrich), anti-HA (clone F-7, sc-7392, Santa Cruz Biotechnology, Dallas, TX, USA), and anti–glyceraldehyde-3-phosphate dehydro-genase (GAPDH) (#CB1001, EMD Millipore) were used as primary antibodies. Anti-rabbit or anti-mouse immunoglobulin G antibody conjugated with horseradish peroxidase was used as a secondary antibody (Thermo Fisher Scientific). Enhanced chemiluminescence reagent, Lumigen ECL Ultra (TMA-6) (Lumigen, Southfield, MI, USA), was used to detect the antigen-antibody complexes.

Immunoprecipitation and HA-p70S6K reporter assaysFor immunoprecipitations, COS-7 cells were transfected with plasmid vectors—AAV empty, AAV-CBA-cTuberin-Myc, pcDNA-hamartin- FLAG (V. Ramesh laboratory), pReceiver-M09/tuberin-Myc (catalog no. EX-Z5884-M09, GeneCopoeia, Rockville, MD, USA), pCMV-Tag3A-Myc-GSK-3 (GSK-3 sequence was cloned into pCMV- Tag3A vector; catalog no. 211173-51, Agilent Technologies, Santa Clara, CA, USA), and pRK5-HA-GST-Rheb1 [catalog no. 19310, Addgene, Watertown, MA, USA; provided by Sancak et al. (59)] using Lipofectamine 2000 (Life Technologies). Cells were lysed with ice-cold phosphate-buffered saline (PBS) (pH 7.4) containing 1% Triton X-100, 2 mM EDTA, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 2 mM sodium vanadate, 10 mM sodium fluoride, and proteinase inhibitors cocktail (Sigma-Aldrich). Lysates were centrifuged at 15,000 rpm for 10 min at 4°C, and pro-tein concentration was measured using the Bradford protein assay

(Bio-Rad). One milligram of lysates was incubated with 2 g of anti-Myc-tag antibody (catalog no. 16286-1-AP, Proteintech, Rosemont, IL, USA) in the presence of Protein A/G Agarose (Santa Cruz Bio-technology) at 4°C overnight. After washing twice with ice-cold modified PBS buffer (pH 7.4) (287 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.05% Triton X-100, and 1 mM EDTA), resins were incubated in 30 l of 0.2 M glycine-HCl buffer (pH 2.5) (Polysciences Inc. Warrington, PA, USA) at RT for 15 min, and then the supernatants were collected and neutralized by adding an equal amount of 1 M tris-HCl (pH 8.0) (Sigma-Aldrich). To increase stringency during the washing, NaCl concentration was increased from 137 to 287 mM in the modified PBS buffer to reduce ionic protein interaction. Eluted immunoprecipitates or whole-cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for Myc-tag (dilution 1:5000) (catalog no. 2276, Cell Signaling Technology), FLAG-tag (1:25,000) (catalog no. F1804, Sigma-Aldrich), and HA-tag (1:3000) (catalog no. sc-7392, Santa Cruz Biotechnology). Anti-mouse antibody conjugated with horseradish peroxidase (Thermo Fisher Scientific) was used as a secondary anti-body (dilution 1:25,000). Enhanced chemiluminescence reagent, Lumigen ECL Ultra (TMA-6) (Lumigen, Southfield, MI, USA), was used to detect the antigen-antibody complexes.

To assess the functional activity of AAV-cTuberin-Myc, we cotransfected HEK293T cells, as previously described with minor modifications (60). Plasmids included HA-tagged p70S6 kinase (HA-p70S6K) (60), which is phosphorylated (pS6K T389) by mTORC1 and was used as a reporter for mTORC1 activation, and Flag-tagged hamartin (Flag-hamartin) (60), along with AAV-cTuberin-Myc. Full-length Flag-tagged tuberin (Flag-tuberin) (60) was used as a positive control, and AAV-GFP was used as a negative control. Transfections were carried out for 48 hours using Lipofectamine 2000. Cell lysates were prepared using radioimmunoprecipitation assay lysis buffer, and immunoblotting was performed, as described (60). Briefly, proteins were separated on a Novex 4 to 12% tris-glycine gradient gel (Life Technologies) followed by transfer to 0.45 M nitrocellulose membrane (Bio-Rad). Antibodies included M2 anti- Flag mouse monoclonal (Sigma-Aldrich), anti-hamartin and anti- pS6K (T389) (Cell Signaling Technology), anti-Myc mouse monoclonal (9E10, University of Iowa Hybridoma Bank), and anti-HA mouse monoclonal (HA.11, BioLegend/Covance, San Diego, CA, USA).

Detection of cTuberin using Western blotHEK293T cells were seeded in a six-well plate (500,000 cells per well) for 24 hours. The cells were then transfected with plasmid DNAs (AAV-null, AAV-GFP, and AAV-cTuberin) at 2.5 g/500,000 cells using Lipofectamine 2000 in Opti-MEM. Six hours later, transfec-tion media was removed and replaced with DMEM (10% FBS and 1% PS), and cells were grown for 72 hours. Cells were washed twice in PBS, and proteins were extracted with protein extraction solution (PRO-PREP, iNtRON Biotechnology, Korea) for 20 min at −20°C. The cell lysates were centrifuged at 14,000g at 4°C. Protein concen-trations of cell lysates were determined using a Bio-Rad protein assay kit. Equal amounts of protein (20 g) were separated using 4 to 12% precast NuPAGE bis-tris SDS-PAGE gels (Invitrogen) and trans-ferred onto nitrocellulose membranes (Thermo Fisher Scientific Inc., Rockford, IL, USA). Membranes were blocked for 1 hour in tris-buffered saline (TBS) with 0.1% Tween 20 and 5% nonfat dry milk, followed by an overnight incubation with primary antibody to tuberin (#3990, 1:1000 dilution, Cell Signaling Technology diluted in


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the same buffer at 4°C). On the next day, the membranes were washed with TBS with 0.1% Tween 20 (three times, 5 min each) followed by incubation with the appropriate horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour at RT. An enhanced chemiluminescence kit (Pierce ECL Western Blotting Substrate, Thermo Fisher Scien-tific, Waltham, MA, USA) was used to detect protein expression. The optical density of each band was determined on Western blots scanned with a G:Box (Syngene, Cambridge, UK).

DNA and RNA extraction, cDNA synthesis, and qPCR for DNABrains and livers were flash-frozen to determine AAV genome bio-distribution and expression of transgene mRNA. Genomic and AAV vector DNA was isolated using the Qiagen DNeasy Blood and Tissue Kit (catalog no. 69504) according to the manufacturer’s instruction. Total RNA was extracted using the Qiagen RNeasy Lipid Tissue Mini Kit (catalog no.74804) and Qiagen RNeasy Mini Kit (catalog no. 74104), with additional on-column deoxyribonuclease (DNase) digestion with the Qiagen RNase-free DNase set (catalog no. 79254) to ensure digestion of AAV-cTuberin genomes. Then, extracted RNA was converted to cDNA using the SuperScript VILO cDNA Synthesis Master Mix (Thermo Fisher Scientific, catalog no. 11754-050), ac-cording to the manufacturer’s protocol. A no-RT set of samples for the AAV-cTuberin group was included to confirm detection of cDNA derived from cTuberin mRNA and not contaminating AAV-cTuberin genomes. Using 50-ng genomic DNA as template, TaqMan qPCR was performed using custom TaqMan probe and primers to 3′ end of cTuberin and c-Myc tag of the transgene expression cassette (for-ward primer, 5′-AGCCAACACCAGGATACGAA-3′; reverse primer, 5′-GCTAATCAGCTTCTGCTCCAC-3′; probe, 5′-FAM- AGCG-GCTGATCTCCTCCGTGG-MGB-3′) (fig. S5). For each sample, a separate qPCR was performed using TaqMan probe and primer sets (Thermo Fisher Scientific, assay ID Mm01180221_g1, gene symbol Gm12070) that detects GAPDH genomic DNA, to ensure equal ge-nomic DNA input for each sample. For each organ/tissue, the AAV vector genome copies for each sample were adjusted by taking into account any differences in GAPDH Ct values using the following formula: (AAV vector genome copies)/(2Ct). The Ct value was calculated as GAPDH Ct value (sample of interest) − average GAPDH Ct value (sample with highest Ct value). Data were expressed as AAV vector genomes per 50 ng of genomic DNA.

Animals and injectionsExperimental research protocols were approved by the Institutional Animal Care and Use Committee for the Massachusetts General Hospital (MGH) following the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals. Experiments were performed on Tsc2c/c-floxed mice [Tsc2-floxed; (61)]. These mice have a normal, healthy life span. In response to Cre recombinase, the Tsc2c/c alleles are converted to null alleles. For vector injections, in the neonatal period (P0 to P3), pups were cryo-anesthetized and injected with 1 to 2 l of viral vector AAV1-CBA-Cre into each ce-rebral lateral ventricle with a glass micropipette (70 to 100 mm in diameter at the tip) using a Narishige IM300 microinjector at a rate of 2.4 psi/s (Narshige International, East Meadow, NY, USA). Mice were then placed on a warming pad and returned to their mothers after regaining normal color and full activity typical of newborn mice. At 3 weeks of age (P21), mice were anesthetized with isoflurane (Baxter Healthcare, Deerfield, IL, USA) inhalation [3.5% isoflurane

in an induction chamber and then maintained anesthetized with 2 to 3% isoflurane and oxygen (1 to 2 liters/min) for the dura-tion of the injection]. AAV vectors were injected retro-orbitally into the vasculature in a volume of 60 l (AAV1 or AAV9) of AAV-cTuberin- Myc using a 0.3-ml insulin syringe over less than 2 min (62) or noninjected.

Body weight measurement and assessment of motor activityEighteen measurements of the body weight of the animals were re-corded from P23 to P50. To assess motor coordination, animals were placed on an automated rotarod apparatus (Harvard Appara-tus, Holliston, MA, USA) using accelerated velocities (4 to 64 rpm over 120 s). Each animal was assessed three times with 5-min rest intervals in each session for nine sessions 3 to 4 days apart. For each assessment, the time ended when the mouse fell off the treadmill or when the time interval elapsed. All functional assessment tests were performed blinded with respect to the mouse genotype.

Detection of the presence of c-Myc using immunocytochemistryHEK293T cells were seeded on coverslip coated with poly-d-lysine (25,000 cells per coverslip) for 24 hours. The cells were then trans-fected with plasmid DNAs (AAV-null, AAV-GFP, and AAV-cTuberin) at 250 ng/25,000 cells using Lipofectamine 2000 in Opti-MEM. Six hours later, transfection media was removed and replaced with DMEM (10% FBS and 1% PS), and cells were grown for 72 hours. The cells were fixed with 4% paraformaldehyde (PFA) (Boston BioProducts, Ashland, MA, USA) for 10 min at RT followed by perme-abilization using 0.01% Triton X-100 (Sigma-Aldrich) in PBS (PBST) for 10 min at RT. The cells were then blocked with 3% bovine serum albumin (BSA) in PBST for 1 hour at RT, followed by overnight incubation with primary antibodies at 4°C [primary antibodies: c-Myc (1:400 dilution; 9E10, Life Technologies)] and GFP (1:400 dilution; A11122, Life Technologies). The cells were then washed three times for 5 min in PBST and incubated with secondary anti-body (goat anti-mouse 488, Jackson ImmunoResearch Laboratories) (1:400 dilution), for 1 hour at RT. The cells were washed three times for 5 min using PBST, mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA). Note that, unfor-tunately, we were not able to detect cMyc in brain sections using several sources of c-Myc antibodies.

Histology and immunohistochemistryThe mouse brains were harvested and subjected for standard his-tological processing as described (14). Five-micrometer sections were stained with hematoxylin and eosin. For frozen sections, adult mice were euthanized using ketamine/xylazine (100:10) (Akorn Inc., Lake Forest, IL, USA) followed by transcardiac perfusion with 1× PBS and 4% PFA in PBS overnight at 4°C, cryo-protected with 25% su-crose in PBS, and embedded in optimal cutting temperature medium (catalog no. 4583, Tissue Teck). Brain sections were prepared in 10-mm coronal sections and were blocked in 10% BSA in 1× PBS + 0.3% Triton X-100 for 1 hour at RT and subsequently incubated with rabbit anti-Ki67 (1:1000; #ab15580, Abcam) or rabbit anti-phospho-S6 ribosomal protein (Ser235/236) (1:400; #2211, Cell Signaling Tech-nology) overnight at 4°C. Following three washes in 0.1× PBS, the sections were incubated with secondary antibody Alexa 555 (1:400; Jackson ImmunoResearch Laboratories) for 1 hour at RT. The sec-tions were then washed three times with 1× PBS and mounted with DAPI mounting medium (Vectashield, #H-1200).


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pS6 puncta analysisWhole mouse brain sections immunostained for pS6 (biological triplicates for each group, three coronal sections per mouse) were imaged using a Nikon Ti2 inverted microscope equipped with W1 Yokogawa Spinning disk scanhead with 50-m pinholes, a Toptica 4 laser launch, and an Andor Zyla 4.2 Plus sCMOS monochrome camera. The slides were mounted on a Nikon linear encoded motorized stage, and the mouse whole brain sections were scanned using Plan Apo 20×/0.8 differential interference contrast (DIC) I objective lens objective lens at 405 nm for DAPI (100-ms exposure) and 561 nm for pS6 staining (100-ms exposure). Signals were col-lected using a Semrock di01-t405/488/568/647 dichroic mirror and Chroma 455/50 or 605/52 nm emission filters. Images were captured using NIS AR 5.02 acquisition software and 12-bit gain four-camera setting. A series of images were captured and stitched together us-ing blending algorithm with 15% overlap among images.

Stitched images were analyzed in Fiji, an open source image pro-cessing package based on ImageJ (63). All images were thresholded within the 80 to 800 tonal range for both DAPI and pS6 staining. An outline was manually drawn to delineate choroid plexuses, ventricles, large empty spots, and meninges from the whole mouse brain sec-tion image. These regions are known to contain significant amounts of autofluorescence and therefore were excluded from downstream analysis. Within the confined region of interests (ROIs), we mea-sured the area for the whole brain section. To identify pS6 puncta size and intensity within them, the thresholded pS6 channel image was converted into eight-bit image and further thresholded within the 70 to 255 tonal range. Subsequently, particle analysis was per-formed to identify any puncta within 5 to 200 m2 and 0.1 to 1.0 circularity parameters. The area for each punctum was measured. These puncta ROIs were then used to identify raw integrated density on original unthresholded 12-bit brain section images. Normalized pS6 puncta number of a brain section was calculated by dividing the total number of pS6 puncta by the brain section area.

Statistical analysisAll analyses of survival curves (Mantel-Cox test and log-rank test) were performed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Flow cytometry analysis on c-Myc–positive cells was analyzed using unpaired t test. Western blot anal-ysis on pS6 and tuberin expression levels in the mouse brain and PS6 puncta parameters were analyzed using unpaired t test. LDH cytotoxicity assay and Western blot analysis on relative levels of S6K T389 phosphorylation were analyzed using one-way analysis of variance (ANOVA) test. P values of



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Acknowledgments: We thank S. McDavitt for editorial assistance, M. F. Lee (Medical Photographer in Pathology Media Laboratory, MGH) for imaging training, M. Zinter (Vector Core, MGH, Charlestown, MA, USA) for AAV vector packaging, and M. Whalen for the use of the rotarod. Funding: This work was supported by DOD Army Grant W81XWH-13-1-0076 (to X.O.B.), NIH R01GM115552 (to M.K.), NIH NIDCD R01DC017117-01A1 (to C.A.M.), NIH NINDS 1R61NS108232 (to X.O.B., C.A.M., and V.R.), and NIH NS109540 (to V.R.). We would like to acknowledge the MGH Vector Core for the production of viral vectors (supported by NIH/NINDS P30NS045776; B.A.T.) and P. M. Llopis, Microscopy Resources on the North Quad (MicRoN), Harvard Medical School, NRB-Longwood, MA, USA. Author contributions: X.O.B., S.P., D.Y., C.A.M., and M.K. conceived and designed the experiments. S.P., P.-S.C., R.L.B., X.Z., and S.K. performed the experiments. S.P., P.-S.C., K.-H.L., and S.K. analyzed the data. S.P., P.-S.C., D.Y., B.A.T., E.A.T., X.Z., R.L.B., R.T.B., D.J.K., A.S.-R., B.G., K.-H.L., V.R., M.K., C.A.M., and X.O.B. wrote and edited the paper. Competing interests: X.O.B., S.P., D.Y., and C.A.M. have filed a provisional patent application for the cTuberin construct. C.A.M. has a financial interest in Chameleon Biosciences Inc., a company developing an enveloped AAV vector platform technology for repeated dosing of systemic gene therapy. X.O.B., V.R., and C.A.M.’s interests are reviewed and managed by MGH and Partners HealthCare in accordance with their competing interest policies. All other authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Plasmid requests can be provided by MGH pending scientific review and a completed material transfer agreement. Requests for the plasmid should be submitted to C.A.M. at [email protected].

Submitted 4 February 2020Accepted 18 November 2020Published 8 January 202110.1126/sciadv.abb1703

Citation: P.-S. Cheah, S. Prabhakar, D. Yellen, R. L. Beauchamp, X. Zhang, S. Kasamatsu, R. T. Bronson, E. A. Thiele, D. J. Kwiatkowski, A. Stemmer-Rachamimov, B. György, K.-H. Ling, M. Kaneki, B. A. Tannous, V. Ramesh, C. A. Maguire, X. O. Breakefield, Gene therapy for tuberous sclerosis complex type 2 in a mouse model by delivery of AAV9 encoding a condensed form of tuberin. Sci. Adv. 7, eabb1703 (2021).


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encoding a condensed form of tuberinGene therapy for tuberous sclerosis complex type 2 in a mouse model by delivery of AAV9

Kaneki, Bakhos A. Tannous, Vijaya Ramesh, Casey A. Maguire and Xandra O. BreakefieldBronson, Elizabeth A. Thiele, David J. Kwiatkowski, Anat Stemmer-Rachamimov, Bence György, King-Hwa Ling, Masao Pike-See Cheah, Shilpa Prabhakar, David Yellen, Roberta L. Beauchamp, Xuan Zhang, Shingo Kasamatsu, Roderick T.

DOI: 10.1126/sciadv.abb1703 (2), eabb1703.7Sci Adv

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