Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder for which there are currently no available therapies. Gain-of-function mutations in the gene encoding superoxide dismutase 1 (SOD1) are responsible for 20% of familial ALS cases. Silencing SOD1 using artificial microRNA has been shown to have therapeutic effects in mouse models of ALS. Here, Borel et al. tested the efficacy and safety of intrathecal delivery of SOD1-targeting artificial microRNAs in nonhuman primates. The therapy efficiently reduced SOD1 protein expression without side effects in macaques. The results suggest that this approach is safe and effective, paving the way for further development of this potential therapy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease caused by degeneration of motor neurons leading to rapidly progressive paralysis. About 10% of cases are caused by gain-of-function mutations that are transmitted as dominant traits. A potential therapy for these cases is to suppress the expression of the mutant gene. Here, we investigated silencing of SOD1, a gene commonly mutated in familial ALS, using an adeno-associated virus (AAV) encoding an artificial microRNA (miRNA) that targeted SOD1. In a superoxide dismutase 1 (SOD1)–mediated mouse model of ALS, we have previously demonstrated that SOD1 silencing delayed disease onset, increased survival time, and reduced muscle loss and motor and respiratory impairments. Here, we describe the preclinical characterization of this approach in cynomolgus macaques (Macaca fascicularis) using an AAV serotype for delivery that has been shown to be safe in clinical trials. We optimized AAV delivery to the spinal cord by preimplantation of a catheter and placement of the subject with head down at 30° during intrathecal infusion. We compared different promoters for the expression of artificial miRNAs directed against mutant SOD1. Results demonstrated efficient delivery and effective silencing of the SOD1 gene in motor neurons. These results support the notion that gene therapy with an artificial miRNA targeting SOD1 is safe and merits further development for the treatment of mutant SOD1-linked ALS.

Because the pathology initiated by mutant SOD1 thus reflects acquired, gain-of-function properties, a potential strategy to treat SOD1-associated ALS is to suppress expression of the SOD1 gene. In recent years, we and others have investigated this strategy in depth using various modalities [reviewed in ( 16 )]. In the present study, we elected to silence SOD1 in cynomolgus macaques using an artificial microRNA (miRNA) targeting multiple SOD1 mutations ( 17 ). We selected a recombinant adeno-associated viral vector serotype rh.10 (rAAVrh.10) because of its excellent central nervous system (CNS) transduction ( 18 ) and safety profile in nonhuman primates. In previous studies in adult SOD1 G93A mice, a model of ALS, we demonstrated that SOD1 silencing profoundly delays disease onset and death and significantly preserved muscle strength and motor and respiratory functions ( 17 ). We further documented that intrathecal delivery of the candidate artificial miRNA in marmoset monkeys (Callithrix jacchus) safely silenced SOD1 in lower motor neurons ( 17 ).

Familial ALS, which represents about 10% of all ALS cases, is inherited as a dominant trait. About 20% of these cases arise from mutations in the gene encoding cytosolic Cu/Zn superoxide dismutase 1 (SOD1) ( 5 ). An estimated 12 to 23% of patients with familial ALS and 1 to 3% of patients with sporadic ALS carry a mutation in this gene; 185 mutations in SOD1 have been identified ( http://alsod.iop.kcl.ac.uk ). Multiple mechanisms have been proposed to explain why mutant SOD1 proteins are neurotoxic, including the observation that mutant SOD1 acquires toxicity via conformational instability, misfolding, and some degree of aggregation ( 6 ). In turn, this activates multiple adverse events (AEs) that include the unfolded protein response ( 7 ), endoplasmic reticulum (ER) stress ( 8 ), mitochondrial damage ( 9 ), heightened cellular excitability ( 10 ), impaired axonal transport ( 11 ), and some elements of apoptotic ( 12 ) and necrotic ( 13 ) cell death. Some data suggest that misfolded mutant SOD1 protein can spread from cell to cell in a prion-like fashion ( 14 ). Additionally, it is proposed that mutant SOD1 can cause toxic misfolding of wild-type SOD1 ( 12 , 15 ).

Amyotrophic lateral sclerosis (ALS) is a devastating, invariably fatal neurological disease caused by degeneration of motor neurons leading to rapidly progressive paralysis. It has an incidence of about 1.5 to 2.5 cases in 100,000 persons in the United States ( 1 , 2 ) and in Europe ( 3 ), or up to about 30,000 new cases of ALS per year in those areas. Survival in ALS is typically 3 to 5 years. There is no cure for ALS, and the U.S. Food and Drug Administration (FDA)–approved treatments extend survival only modestly ( 4 ).

RESULTS

Optimized intrathecal rAAV delivery at the lumbar spinal cord section is well tolerated and leads to widespread vector biodistribution and green fluorescent protein expression in primate spinal cord and brain To silence SOD1, we used a previously developed artificial miRNA targeting multiple SOD1 mutations driven by polymerase II (pol II) or polymerase III (pol III) promoters, and delivered with a recombinant AAV serotype rh.10 (17). Here, we chose to work with large nonhuman primates, the cynomolgus macaques (M. fascicularis). To minimize intersubject variability and therefore allow for robust testing of the preclinical candidate, macaques for study were of the same gender and close in age and body weight (table S1). Ten days before infusing our therapy, a polyethylene-lined polyurethane catheter [outer diameter (OD), 1.0 mm; inner diameter (ID), 0.38 mm] equipped with a MIN-LOVOL subcutaneous titanium access port for infusions was placed via radiographic guidance; the catheter entered the intrathecal space and extended to the low thoracic region (Fig. 1A). After 10 days, the entry site was well healed, permitting slow infusion without backflow. Previous work showed that placing the subject in the head-down, Trendelenburg position improved CNS transduction (19). Accordingly, our animals were restrained in a prone position with the restraint table tilted about 30° head-down (Fig. 1B) for the duration of the dosing procedure. The animals were not anesthetized during this procedure; to allow better monitoring during the infusion, they were trained to be restrained in this position. Fig. 1 Optimized intrathecal rAAV delivery at the lumbar spinal cord section is well tolerated and leads to widespread vector biodistribution and GFP expression in primate spinal cord and brain. (A) Myelogram showing the magnified thoracolumbar region with the catheter tip, implanted at least 10 days before the vector delivery. (B) The subject is restrained and placed in the Trendelenburg position, at a 30° angle, for vector infusion. (C) Vector biodistribution throughout the CNS and peripheral organs determined by droplet digital polymerase chain reaction (ddPCR). CB-miR-SOD1 (n = 4); H1-miR-SOD1-GFP (n = 4); U6-miR-SOD1-GFP (n = 4). Error is represented as SD. (D and F) Detection of GFP protein expression in the cervical spinal cord (D) and brain (F). (E and G) GFP expression in lower motor neurons (E) and pyramidal neurons in the Brodmann area 4 in brain (G) of primates injected with the preclinical H1 vector. CSC, cervical spinal cord; LSC, lumbar spinal cord; TSC, thoracic spinal cord; CB, clinical CB vector; H1, preclinical H1 vector; U6, preclinical U6 vector. We designed the study to also compare the use of both pol II (CB) and pol III (H1 and U6) promoters driving the expression of the artificial miRNA against SOD1 (miR-SOD1). The vector was administered as two 2.5-ml infusions through the intrathecal lumbar (IT-L) port/catheter system, the second infusion being separated from the first one by at least 6 hours to allow for cerebrospinal fluid (CSF) turnover. The animals were split into four groups, and each group received either no vector, a CB-miR-SOD1 [clinical CB, without green fluorescent protein (GFP)] vector, an H1-miR-SOD1 (preclinical H1, included GFP) vector, or a U6-miR-SOD1 (preclinical U6, included GFP) vector, as described in table S1. The procedure was well tolerated during both the infusion and the postprocedure period, during which experienced animal caretakers frequently monitored the animals. No gross adverse side effects were observed during the course of the postprocedure monitoring; the treated animals demonstrated normal behavior and food intake. This injection protocol achieves widespread transduction in the spinal cord, brain, and peripheral organs, as shown by analysis of the vector biodistribution (Fig. 1C); the numbers of vector genome copies per diploid genome (vg/dg) ranged from 10 to 100 in the spinal cord. The vector biodistribution was then confirmed at the protein level with analysis of the GFP expression in the spinal cord and in the brain. Our results show transduction in the spinal cord of both the anterior and posterior horns (Fig. 1, D and E, and fig. S1) and widespread transduction of the brain. Coronal brain sections show GFP staining patterns consistent with corticospinal tracts emanating from the cortex and traversing the cerebral peduncle, as well as the pontocerebellar fibers in the pons and oculomotor nucleus in the midbrain. We also observed GFP-positive staining of cortical layer V pyramidal cells in the primary motor cortex (Brodmann area 4) (Fig. 1, F and G, and figs. S2 and S3).

Delivery of preclinical H1 and U6 vectors leads to elevation of liver transaminases Although the animals showed no behavioral evidence of toxicity from this infusion, we observed an elevation in liver transaminases outside of the normal range on day 22 in animals treated with both H1-miR-SOD1 and U6-miR-SOD1 vectors carrying the GFP sequence. Conversely, animals treated with CB-miR-SOD1 (without GFP) did not show liver transaminase increase. This was not present at day 3 after injection (necropsy samples; Fig. 2, A and B, and table S2). In previous clinical studies with AAV targeting the liver, an increase in serum transaminases has been associated with the reactivation of a response of cytotoxic T lymphocytes (CTLs) to AAV capsid (20, 21). This seemed unlikely in our study, because liver function elevation was observed in only two of the three groups, yet all three cohorts had received the same AAV capsids at equivalent doses. We therefore formulated the alternative hypotheses that the liver toxicity reflected either (i) the presence of the GFP protein or (ii) the high expression of miR-SOD1 precursors in the two cohorts with pol III promoters (H1 and U6). Conceivably, the miRNA precursors might saturate the endogenous miRNA pathway. To discriminate between these two hypotheses, we analyzed the cellular immune response to GFP protein with a standard interferon γ (IFN-γ)–ELISpot assay. Spleens were harvested at necropsy, and splenocytes were isolated after enzymatic dissociation (22 days after dosing). These cells were expanded for 6 days before running the ELISpot assay. For the IFN-γ–ELISpot assay, splenocytes were stimulated for 48 hours with overlapping peptides spanning the entire GFP protein sequence and divided into three pools. Two of eight animals developed a cellular immune response to the GFP protein detectable by ELISpot (fig. S4). Fig. 2 Delivery of preclinical H1 and U6 vectors leads to elevation of liver transaminases. (A and B) Aspartate aminotransferase (AST) (A) and alanine aminotransferase (ALT) (B) quantification in serum after vector injection. (C) SOD1 expression in the liver determined by ddPCR and normalized to HPRT. Unpaired two-tailed t test; CB versus H1, P ≤ 0.0045 and CB versus U6, P ≤ 0.0447. (D) Mature miR-SOD1 expression in the liver quantified by ddPCR and normalized to U47. (E) Hepatocellular pathology score [two-tailed analysis of variance (ANOVA) CB versus H1 and U6, P ≤ 0.0001] and (F) Ki67 score (two-tailed ANOVA; CB versus H1 and U6, P ≤ 0.0178). Error is represented as SEM. Control, uninjected animals (n = 3); CB (n = 4); H1 (n = 4); U6 (n = 4). *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001. An in-depth analysis of the liver phenotype was then performed, both to identify the pathological basis for the observed liver toxicity and to assess efficacy of SOD1 suppression. Silencing of SOD1 in the liver was determined by ddPCR (Fig. 2C). Silencing was more complete with the pol II (CB) promoter than with the pol III (H1 and U6) promoters; as compared to untreated animals, the mean relative SOD1 expression was 0.08 in the CB group, 0.54 in the H1 group, and 0.33 in the U6 group (H1 versus CB, P ≤ 0.0045; U6 versus CB, P ≤ 0.0447). Next, mature miR-SOD1 molecules were quantified by ddPCR (Fig. 2D). Mature miR-SOD1 was detected in the liver of the animals treated with the pol II (CB) promoter and with the pol III (H1 and U6) promoters; compared to controls, the mean relative miR-SOD1 expression was 22.44 in the CB group, 0.19 in the H1 group, and 0.36 in the U6 group (statistical analysis is provided in table S3). Liver samples were subsequently subjected to a blind analysis by an experienced pathologist. Scoring of the liver histopathology (severity scores for portal mononuclear infiltrate, hepatocyte cell death, and hepatocyte regeneration) revealed a significant increase in the scoring (reflecting increased pathology) in the two pol III–treated groups compared to the untreated control animals (Fig. 2E; P ≤ 0.0001). Additionally, a Ki67 staining was performed and Ki67-positive cells were counted (Fig. 2F). The Ki67 immunostaining score, which reflects the degree of cellular proliferation, was significantly increased in the H1 and U6 groups compared with untreated controls (P ≤ 0.0178). Hepatocellular damage and Ki67 scores were positively correlated (P ≤ 0.0001; fig. S5). Although the pathology results confirmed the liver injury, they did not elucidate which of the two hypothetical causes for the liver toxicity was driving this process (GFP toxicity or pol III–mediated toxicity) (22, 23). However, quantification of the expression of miR-122, the most abundant endogenous miRNA in the liver, revealed stable miRNA expression among treatment groups (fig. S6), suggesting that the toxicity is not caused by saturation of the endogenous miRNA machinery due to high expression of miR-SOD1 from the pol III promoters.

SOD1 is profoundly and reproducibly silenced in motor neurons The mature miRNA, miR-SOD1, was quantified in the spinal cord at three sections: lumbar (LSC), thoracic (TSC), and cervical (CSC) (Fig. 3A). The SD within each group was limited, suggesting that there was good technical reproducibility. The results show that the quantity of miR-SOD1 increased with the relative strength of the promoter (CB < H1 < U6) (Fig. 3A). Ninefold higher miR-SOD1 expression levels were detected in the H1 group compared with the CB group (P ≤ 0.0003), and a 15-fold increase was found for the U6 group compared with the CB (P ≤ 0.0002). Last, to precisely assess silencing in the motor neurons, which are the primary cellular target for this therapy, an average of 400 cells were microdissected using laser capture. The resulting expression profile in motor neurons demonstrated significant silencing (Fig. 3B and table S3). Overall, the extent of the silencing positively correlated with the strength of the promoter (CB < H1-U6). Fig. 3 SOD1 is profoundly and reproducibly silenced in motor neurons. (A) Relative mature miR-SOD1 expression in the spinal cord determined by ddPCR and normalized to U47. Unpaired two-tailed t test; control TSC versus CB TSC, P ≤ 0.0188; control TSC versus H1 TSC, P ≤ 0.0036; control LSC versus H1 LSC, P ≤ 0.0034; control CSC versus U6 CSC, P ≤ 0.0105; control TSC versus U6 TSC, P ≤ 0.0162. (B) Relative SOD1 expression in laser-capture microdissected motor neurons quantified by ddPCR and normalized by HPRT. Unpaired two-tailed t test; control LSC versus CB LSC, P ≤ 0.0014; control CSC versus H1 CSC, P ≤ 0.0034; control TSC versus H1 TSC, P ≤ 0.0009; control LSC versus H1 LSC, P ≤ 0.0001; control CSC versus U6 CSC, P ≤ 0.0097; control TSC versus U6 TSC, P ≤ 0.0007; control LSC versus U6 LSC, P ≤ 0.0002. Error is represented as SEM. (C and D) GFP and SOD1 immunostaining in the lumbar spinal cord of control and vector-injected animals. DAPI, 4′,6-diamidino-2-phenylindole. Control, uninjected animals (n = 3); CB (n = 4); H1 (n = 4); U6 (n = 4); Scale bars, 50 μm. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Our results show a 1.7-fold (P ≤ 0.0053) and 1.6-fold (P ≤ 0.0074) decrease in the H1 and U6 groups compared with the CB group, respectively, in the CSC, a 2.1-fold (P ≤ 0.0152) and 2.5-fold (P ≤ 0.0052) decrease in the TSC, and a 3.6-fold (P ≤ 0.0033) decrease in H1 versus CB in the LSC. There was no difference between the CB and the U6 group in the LSC. Overall, differences between control versus H1 and control versus U6 throughout the spinal cord were statistically significant, and differences between control versus CB were statistically significant in the LSC (table S3). SOD1 silencing was generally lower in the cervical section (CSC), intermediate in the thoracic section (TSC), and higher in the lumbar section (LSC), with up to 93% silencing in the LSC of the H1 group (table S3). These results were then confirmed at the protein level. LSC sections were stained with anti-GFP and anti-SOD1 antibodies. As expected, an uninjected control primate shows no GFP expression but strong SOD1 expression in motor neurons (Fig. 3C). Animals injected with the preclinical H1 vector (that included GFP) show GFP expression in most but not all motor neurons, confirming that rAAVrh.10 targets motor neurons. The GFP and SOD1 co-staining shows that the motor neurons expressing the GFP protein are not expressing SOD1, whereas GFP-negative motor neurons in the same tissue section do have SOD1 protein (Fig. 3D).

GFP-devoid clinical constructs are not linked to elevation of liver transaminases A second experiment was subsequently carried out using the same delivery protocol. The goals of this experiment were to test whether the GFP was indeed the cause of the aforementioned mild liver toxicity and to demonstrate safety of the GFP-devoid clinical vectors. This experiment also afforded an opportunity to assess the impact of preexisting neutralizing antibodies (NAbs) to AAV in the periphery on intrathecal AAV injection. The animals were split into four groups; each group received either no vector, an H1-miR-SOD1 (clinical H1) vector (studied at day 43), an H1-miR-SOD1 (clinical H1) vector (studied at day 92), or a CB-miR-SOD1 (clinical CB) vector (also evaluated at day 92), as described in table S4. To determine the impact of preexisting NAb on vector biodistribution, we noted that three of seven animals selected in the H1-miR-SOD1 (short-term) group were seropositive for AAVrh.10 before dosing (table S5). Analysis of the CNS and peripheral organs revealed a widespread biodistribution of the virus in the spinal cord, brain, and peripheral organs (Fig. 4A). These results show that there was neither a difference in the biodistribution of the AAV in the CNS in the group with preexisting NAbs (Table 1) nor a reduction of vector genomes per cell in the spinal cord (table S6). It should be noted that the first study ended after only 22 days, whereas the second study had day 43 and day 92 end points. To verify that there is no loss of vector genome due to a clearance of extracellular AAV genomes between day 22 and days 43 and 90 in transduced motor neurons, which are the main target in this study, we performed a biodistribution on 200 laser-captured motor neurons from animals injected with preclinical or clinical H1 vectors and euthanized at days 22 and 92, respectively. The results showed no difference in viral genome copy number per diploid genome in these cells at the two time points (table S7). Fig. 4 GFP-devoid clinical constructs are not linked to elevation of liver transaminases. (A) Vector biodistribution throughout the CNS and peripheral organs determined by ddPCR. Error is represented as SD. (B and C) AST (B) and ALT (C) expression in serum after vector injection. (D) SOD1 silencing in the liver determined by ddPCR and normalized to HPRT. Error is represented as SEM. Control, uninjected animals (n = 3); CB (n = 3); H1 [n = 7 (43 days) and n = 4 (92 days)]. The numbers in parentheses below the vector name denote the number of days at end point for each group. Table 1 Preexisting NAbs have no impact on spinal cord transduction and silencing. MN, motor neurons; no NAb, low NAb titer before dosing; NAb+, animal positive for NAb before dosing. View this table: In this second primate study, blood was sampled frequently (at days 8, 15, 22, 29, and 43) to allow for close monitoring of clinical chemistry parameters. Liver transaminases remained within the normal range and were stable throughout the course of the study (Fig. 4, B and C). These results suggest that the previously observed liver toxicity was not linked to the pol III promoters but was likely caused by the GFP (CB versus H1; first study, day 22: ALT, P ≤ 0.0001; AST, P ≤ 0.0001; second study, days 22, 29, and 43: ALT, not significant; AST, not significant). Analysis of liver SOD1 expression is shown in Fig. 4D.

Clinical constructs led to reproducible SOD1 silencing To assess silencing of SOD1 in motor neurons, cells were laser captured and used to isolate RNA and subsequently quantify SOD1 expression (Fig. 5, A to C). At 92 days after injection, silencing exceeded 30% in the CB group and 60% in the H1 group in the lumbar section (Fig. 5A). An intermediate degree of silencing was observed in the thoracic section (Fig. 5B), and in the cervical section, the difference between groups was reduced, with about 35% silencing in the CB group and about 45% silencing in the H1 group (Fig. 5C). The amount of mature miR-SOD1 was detected only in the treated groups (Fig. 5, D to F) and was fairly stable along the spinal cord in the lumbar (Fig. 5D), thoracic (Fig. 5E), and cervical (Fig. 5F) sections. The difference in miR-SOD1 expression between pol II and pol III groups at 92 days is fivefold in the lumbar section (P ≤ 0.0155, table S8), and there was no difference further away from the injection site (thoracic and cervical). SOD1 silencing was confirmed by branched fluorescence in situ hybridization (FISH) (RNAscope) assay where transduced motor neurons show an important decrease in SOD1 signal (Fig. 5G). Fig. 5 Clinical constructs led to reproducible SOD1 silencing. SOD1 silencing measured by ddPCR in laser-capture microdissected motor neurons at the lumbar [(A) unpaired two-tailed t test; control versus H1 (43), P ≤ 0.0001; control versus H1 (92), P ≤ 0.0008; CB (92) versus H1 (92), P ≤ 0.0284], thoracic [(B) unpaired two-tailed t test; control versus H1 (43), P ≤ 0.0045; control versus H1 (92), P ≤ 0.0087], and cervical [(C) unpaired two-tailed t test; control versus CB P ≤ 0.0457, control versus H1 (43) P ≤ 0.0067, control versus H1 (92) P ≤ 0.0099] section of the spinal cord. Mature miR-SOD1 detection in primates by ddPCR at the lumbar [(D) unpaired two-tailed t test; control versus CB, P ≤ 0.0131; control versus H1 (92), P ≤ 0.0038; CB (92) versus H1 (92), P ≤ 0.0155], thoracic [(E) unpaired two-tailed t test; control versus CB, P ≤ 0.0094], and cervical (F) section of the spinal cord. Control, uninjected animals (n = 3); CB (n = 3); H1 [n = 7 (43 days) and n = 4 (92 days)]. The numbers in parentheses below the vector name denote the number of days at end point for each group. (G) Motor neurons stained by branched FISH (RNAscope) assay, ChAT (choline acetyltransferase; red), vector genomes (vg, green), and SOD1 (yellow). Scale bars, 10 μm. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Clinical constructs are safe Last, small RNA sequencing (RNA-seq) was performed to more precisely examine the small RNA species generated by the vector-derived artificial miRNA at 92 days after treatment. Depending on the flanking sequences used (here that of hsa-miR-155), miRNA processing can vary. Important parameters for safety include the abundance of the predicted mature miRNA over alternate species (which should be high, hence avoiding potential off-targeting), as well as the guide/passenger strand ratio (which should be high as well, again limiting the risk of off-targeting). Although preliminary work using these flanking sequences to silence another target demonstrated precise processing (24), we sought to confirm these findings in the context of miR-SOD1 in an in vivo setting by small RNA-seq. Alignment of all reads mapping to the miR-SOD1 precursor showed that the most abundant species for both the guide and the passenger strand are the predicted sequences (Fig. 6A). In the case of the guide strand, the predicted species was 100-fold more abundant than alternate species (Fig. 6A). Moreover, the predicted/most abundant mature guide was about 100-fold more abundant than the predicted/most abundant mature passenger (Fig. 6A). Quantification of mature miR-SOD1 (guide strand) showed that, in vivo, the H1 promoter expressed about fivefold more miR-SOD1 than the CB promoter (Fig. 6B, P ≤ 0.0030). Fig. 6 Clinical constructs are safe. (A) Small RNA-seq catalog of all the artificial miRNA species generated in vivo by alternative processing of the vector-encoded construct. CB, n = 3; H1, n = 4. (B) Comparison of the expression of mature miR-SOD1 generated in vivo by the CB or the H1 vector. CB, n = 3; H1, n = 4. Unpaired two-tailed t test; CB versus H1, P ≤ 0.003. Analysis of potential off-target effects by ddPCR on control animals (n = 3 to 4) and animals injected with H1 clinical vector (n = 4, 43 days) at the cervical (C) and lumbar (D) sections of the spinal cord and in the liver (E). Each gene was normalized to HPRT. Error is represented as SEM. Unpaired t test; control versus H1. We then analyzed potential off-target effects in the group injected with the clinical H1 vector and euthanized at day 43 after injection. We identified four potential off-target genes on the basis of high sequence identity (52 to 71%) between the 21-nucleotide-long mature miR-SOD1 and the messenger RNA of genes cataloged in the M. fascicularis RefSeq_RNA database (table S9). We analyzed the expression levels of these genes (Fig. 6, C to E). This analysis was performed on the CSC (Fig. 6C) and LSC (Fig. 6D), as well as on the liver (Fig. 6E) (because the liver demonstrated the highest vector copy number per cell in periphery). Our results show no off-target silencing in terms of mRNA relative expression for each target comparing uninjected animals to animals injected with the clinical H1 vector.