First-in-human AAV Gene Therapy for Tay-Sachs Disease

27 Tay-Sachs Disease (TSD) is an inherited neurological disorder caused by deficiency of 28 hexosaminidase A (HexA). Preclinical work demonstrated safety and efficacy of CNS gene 29 therapy using AAVrh8-HEXA/HEXB. Here we describe an expanded access trial in two patients 30 with infantile TSD (IND 18225). 31 Case TSD-001 demonstrated neurodevelopmental regression by 8 months of age and severe 32 seizures by 1 year was treated at 30 months. An equimolar mix of AAVrh8-HEXA and AAVrh8- 33 HEXB (now AXO-AAV-GM2) was administered intrathecally (IT), with 75% of the dose (1x10 14 vg) 34 delivered to the cisterna magna and 25% at the thoraco-lumbar junction. The second patient 35 (TSD-002) was treated at 7 months of age with 4∙2x10 13 vg by a combination of bilateral thalamic 36 (0∙18 mL; 1∙5x10 12 vg per thalamus), and IT infusion (3∙9x10 13 vg). Both patients underwent 37 immunosuppression with sirolimus, corticosteroids, and rituximab. 38 Injection procedures were well tolerated and have shown no vector-related adverse events to 39 date. CSF HexA activity nearly doubled from baseline and remained stable. In TSD-002 (now 40 16 months of age), MRI showed stabilization of disease by 3 months post-injection; there now 41 appeared to temporarily deviate from the natural history of infantile TSD but declined again 6 42 months post-treatment. TSD-001 (now 4.5 years of age remains seizure-free on the same anti- 43 convulsant therapy as pre-therapy, but TSD-002 developed seizures between 13 and 17 months 44 post-treatment (by 2 years of age). AXO-AAV-GM2 by IT and thalamic HexA activity in CSF and ongoing myelination was apparent in the younger patient treated at an early symptomatic stage. map (FA) (RD)


Radiologic Assessments 144
Post-injection MRI images showed successful thalamic targeting in TSD-002, without any 145 detectable deleterious effects ( Figure 3A). Fluid signal averaged 474 mm 3 per thalamus, ~2•6 146 times the infused volume. At the time of treatment, TSD-001 had advanced cortical atrophy, mild 147 ventricular enlargement and diffusely affected white matter ( Figure 3B), but after treatment no 148 further deterioration was apparent on MRI. At baseline, TSD-002 MRI was suggestive of mild 149 dysmyelination that appeared unchanged at 3 months ( Figure 3B). At 6 months, areas of 150 increased myelination were noted in several sites, including the posterior corpus callosum. DTI 151 showed minimal changes over time, except in the anterior corpus callosum of TSD-001 ( Figure  152 3C, supplementary Figure S1). Volumetric analyses from TSD-001 suggested stabilization of 153 corpus callosum volumes after treatment. Total brain volume of TSD-002 increased over the 6-7 month period, a finding also noted in individual brain structures ( Figure  TSD-002. TSD-001 remained stable with a score of ~20 ( Figure 2E). Presently, TSD-001 is 4 159 years and 6 months old has been seizure-free since treatment on the same anticonvulsant 160 therapy (Keppra) and non-adjusted dose that was in place pre-therapy when seizures were active. 161 Electroencephalograms, auditory brainstem responses, and retinal exams did not change 162 significantly through the first 6 months after therapy, after which time data were not available due 163 to health-related travel restrictions. 164 165 TSD-002's score remained ~60 until 6 months, when it decreased to 52. At 13 months of age, 166 TSD-002 was still sitting, an important milestone for infantile TSD 1 , but with some truncal stability 167 loss and exaggerated startle response. At 16 months of age, TSD-002 could sit for 5 seconds, 168 retained visual tracking and response to auditory stimuli, but this was ability was lost by 24 months 169 of age. Seizures developed by 24 months of age (17 months post-therapy). As with the first 170 patient, electroencephalograms, auditory brainstem responses, and retinal exams did not change 171 significantly through the first 6 months after therapy, after which time data were not available. 172

Conclusions 174
This is the first in human test of AAV gene therapy in TSD; this trial achieved a mild increment in 175 CSF HEXA activity, showed good general safety in both patients, and demonstrated feasibility of 176 bilateral thalamic injections in a 7-month-old infant. There were no vector related SAEs, and mild 177 AEs after treatment were judged to be related to procedural aspects of the protocol, including a 178 brief, mild post-operative fever. Transaminase levels remained within the historical range 179 8 reported for TSD 25 . No anti-transgene immune responses were observed, and anti-capsid 180 immune responses were short-lived and mild. 181 Post-treatment CSF HexA activity in the range observed here could alter the trajectory of TSD, 182 since patients with 0.5% of normal HexA activity exhibit a late infantile/ juvenile phenotype 26 . 183 HexA levels in CSF may not accurately represent brain tissue activity, but nonetheless MRI of 184 TSD-002 showed some degree of disease burden stabilization and increased myelination in 185 several structures, in agreement with some attenuation of clinical disease progression. If 186 sustained, this would represent a meaningful benefit when compared to siblings and natural 187 course of infantile TSD. This impact occurred in TSD-002 patient despite receiving half the dose 188 of TSD-001, pointing to the value of early treatment. A retrospective study of infantile Tay-Sachs 189 disease showed that 98% of patients developed seizures with an onset at 16.8 ± 5.5 months of 190 age that are often refractory to antiepileptic drugs 1 . The observation that TSD-001 has remained 191 free of seizures at 4.5 years of age (24 months post-therapy), in spite of continuing the same anti-192 convulsant therapy (Keppra) at a dose which had not been adjusted since gene therapy, suggests 193 a positive benefit of the gene therapy intervention. Although this is encouraging, this effect did 194 not persist in TSD-002, where seizures were noted by 24 months of age (17 months after therapy). 195 This is the first report of intrathalamic gene therapy in patients. The technique holds potential for 196 treatment of other diseases, because axonal transport enables global CNS distribution 21,22 . The 197 thalamus is a high-risk target because of its critical functions as an integration and relay center of 198 the cerebral cortex. Therefore, to minimize risk to the patient, the thalamic injection volume in 199 TSD-002 (180 µL) was roughly equivalent to that safely injected in non-human primates (150 µL; 200 data not shown). The dose/volume used corresponds to ~1/10 of that projected to be most 201 efficacious in pre-clinical experiments.
Since the thalamic volume is estimated at ~4,500 202 microliters 27 , and infusate spread was ~474 microliters, a substantial increase in dose/volume will 203 be needed to achieve maximal therapeutic benefit. Animal studies showed a 10-fold higher 204 thalamic injection volume is safe; however, thalamic injury in patients is of concern. Since TSD 9 is a fatal disease, risk associated with injecting larger volumes is warranted and necessary to 206 achieve transformative therapeutic outcomes. 207 In summary, expanded access use of AXO-AAV-GM2 was safe after intrathecal and bilateral 208 thalamic delivery, and accompanied by a modest increase in CSF HEXA activity. Clinical findings 209 in TSD-002 who was treated at a younger age, suggest a possible deviation from the natural 210 history of TSD despite receiving a lower dose than indicated by preclinical experiments. This        Figure S1: Diffusion tensor imaging (DTI) results. A) Mean diffusivity (MD) maps of TSD-002 brain indicating regions of interest (ROI) for calculation of DTI parameters. MD quantifications over time are displayed below the images for various ROIs in the brain for both TSD-001 and TSD-002. B) Axial diffusivity (AD) and C) radial diffusivity (RD) measurements are also shown for the same brain ROIs over time for both patients. In TSD-002 both optic tracts show stable FA values and increased average diffusivity values. AD values remain stable even when FA decreases, as shown in the anterior corpus callosum for TD-001. Figure S2: volumetric calculations of lateral ventricular size and size of the lentiform nucleus. TSD-001 has larger ventricles consistent with cortical atrophy whereas the relevance of TSD-002 ventricular size remains unclear and could be associated with normal brain development.

Hex A enzyme activity
Cerebral spinal fluid or serum from patients and non-TSD controls were used either neat or and rabbit polyclonal anti-human prealbumin (1:500; A0002, Dako). All primary antibodies were incubated at 4˚C for 16-18 hr. Detection was performed by chemiluminescence using Clarity Western ECL Substrate (Bio-Rad) and images were acquired using the ChemiDoc system (Bio-Rad).
Mass Spectrometry quantification of GM2 ganglioside in CSF C-MS/MS analysis was conducted on a Shimadzu Prominence UFLC system coupled with an Applied Biosystems/MDS Sciex 000QTRAP mass spectrometer using multiple reaction monitoring (MRM) and two Valco switching valves. The CSF samples were injected directly to the LC-MS/MS system. A quality control sample was prepared by pooling 20 mL from each study sample and injected every three study samples to monitor the instrument performance.
The relative quantification data were measured as peak areas of analytes and reported as total counts. All the samples were analyzed in one batch so that relative comparisons could be made accurately without run-to-run variation. The coefficient of variation of each lipid in the quality control sample was less than 15%, indicating that the mass spectrometric response was reproducible within the batch.
Cells were washed and developed with Galactor-Star kit (Applied Biosystems). Luminescence was measured with a luminometer. The NAb titers are expressed as the highest dilution that inhibited β-galactosidase expression by at least 50% compared to a negative mouse serum control.

Volumetric analysis
Amira (V2019.1, Thermo Fisher Scientific, Waltham, MA) was used to perform volume calculations of the different brain regions at each time point for each patient. A combination of 3D T1-and 3D T2-weighted MR images were used for segmenting the following regions: whole brain (without ventricles), cerebellum, thalamus, corpus callosum, caudate, ventricles, and lentiform nucleus. For the whole brain and ventricle regions, a semi-automated technique using a combination of thresholding and edge detection was used to capture the respective regions in each coronal slice and manual corrections were performed thereafter before rendering the 2D slices into a 3D volume. The remaining structures were segmented manually due to isointense signal of the structures with surrounding tissue. All segmentations were performed using both the axial and sagittal orientations at the same time while carefully paying attention to tissue boundaries. All segmentations were verified using age-appropriate atlases under the guidance of an experienced neuroradiologist (SR).

DTI analysis
DTI analysis was performed using DSI Studio (http://dsi-studio.labsolver.org). For each patient at each time point, fractional anisotropy (FA), axial diffusivity (AD), mean diffusivity (MD), and radial diffusivity (RD) maps were generated to visualize the changes in the brain over time. To further quantify the changes in sub-regional structures, the following regions of interest were selected: anterior and posterior corpus callosum, dorsal brainstem, left and right centrum semiovale, superior cerebellar peduncle, left and right proximal occipital optic tractus, and left and right distal occipital optic tractus. FA, MD, AD, and RD quantification were made for each of the aforementioned structures and tracked over time for each patient. Figure 1 Longitudinal safety and immunological studies. Serum levels of liver enzymes A) alanine aminotransferase (ALT) and B) aspartate aminotransferase (AST) enzymes in TSD-001 and TSD-002 patients before treatment (Pre-TX), weeks 1, 2, and 3 (W1, 2, 3) and months 1, 2, 3 and 6 (M1, 2, 3, 6).

Figures
Shaded bars in A) and B) represent the normal interval for both assays. Dashed line in B) indicates the maximum value reported in TSD patients25. C) Total anti-AAVrh8 IgG in serum quanti ed by ELISA. In C) the gray squares and lines represent anti-capsid IgG levels in serum of an ALS patient treated by IT AAV gene therapy. D) Neutralizing antibody titers to AAVrh8 capsid quanti ed by a transduction assay. Peripheral blood mononuclear cells (PBMCs) were isolated from TSD-001 and TSD-002 patients before treatment and at various time points thereafter to assess T-cell responses by interferon gamma ELISpot assays using stimulation with pools of overlapping peptides spanning E, F) AAVrh8 VP1, or G, H) human HEXA and HEXB proteins. Unstimulated or CD3/CD28-stimulated PBMCs were included in all assays as negative and positive controls. A-D) TSD-001 data is represented by blue circles and connecting lines, while TSD-002 data is represented by red triangles and connecting lines. Longitudinal biochemical and neurological function outcome measures. HexA activity was measured in A) cerebral spinal uid (CSF) and B) serum of TSD-001 (blue bars) and TSD-002 (red bars) patients and non-TSD individuals (gray bars). CSF was collected on treatment day prior to infusion (D0), months 3 and 6 (M3, 6). Serum was collected at weeks 1, 2, 3 (W1-3), months 2, 3, and 6 (M2, M3, M6). The increase in HexA activity levels in CSF and serum post treatment was statistically signi cant using a mixed effects model to adjust for repeated measures within individual. C) Mass spectrometry quanti cation of GM2 ganglioside species in CSF at day 0, months 3 and 6. TSD-001 data is represented by blue circles and connecting lines. TSD-002 data is represented by red triangles and connecting lines. D) Western blot analysis of HEXA and HEXB protein expression in CSF of TSD-001 and TSD-002 patients at day 0 (D0), months 3 (M3) and 6 (M6). Transthyretin (TTR), which is the most abundant protein in CSF, was used as a loading control. Arrowheads indicate the location of the bands corresponding to the HEXA andHEXB proteins. E) CHOP-INTEND scores for patients TSD-001 and TSD-002 from 2 days prior to treatment until 6 months or 12 months post-treatment. and diffusely affected white matter over the 6 month period. Color map (CM) of the fractional anisotropy (FA) and radial diffusivity (RD) suggest a combination of hypomyelination and myelin loss. The pretreatment MRI for TSD-002 shows incomplete myelination during infancy as posterior limbs of the internal capsule were not myelinated and lacked parietal and occipital myelination. After treatment, the degree of myelination, as shown by the darkening on T2 imaging (B) and FA metrics (C), suggest stabilization over time and potential improvement in the posterior corpus callosum.