Mutation Identification

Whole-genome sequencing was undertaken to search for the missing second mutation, which was hypothesized to be a noncoding mutation or a structural variant missed by standard clinical sequencing. Careful inspection of sequencing data revealed a cluster of chimeric reads deep in MFSD8 intron 6 (Figure 1B), detected in both the proband and the mother (Fig. S2A). Chimeric reads in this cluster were fused either to unmapped poly-T sequences on one end or to unmapped hexameric repeat sequences (AGAGGG) on the other end, which suggested the presence of a DNA insertion beginning and ending with these motifs. Chimeric breakpoints were offset by 14 bp, suggesting duplication of an endogenous target sequence (Figure 1B).

We deduced that these features were consistent with the insertion of an SVA (SINE–VNTR–Alu) retrotransposon.4 Indeed, amplification by polymerase chain reaction (PCR) across the breakpoints revealed an approximately 2-kb insertion in the proband and the mother that was confirmed by RepeatMasker analysis to be an SVA retrotransposon. The segregation of the variant within this family was confirmed in a Clinical Laboratory Improvement Amendments–certified laboratory to match recessive inheritance (the proband is compound heterozygous for the SVA insertion and c.1102G→C, and the parents are heterozygous carriers). The SVA insertion was also correctly detected by automated analysis with the Tea algorithm.5 This insertion has not been reported in the literature and does not appear in dbRIP, a database of retrotransposon insertions in the human population.6 Its absence was also confirmed in more than 800 persons by Tea analysis of publicly available human whole-genome sequencing data sets. (Additional details of the results of these analyses are provided in Figs. S2 through S4.)

Because SVA insertions have been reported to modulate splicing of nearby genes,7 we examined splicing patterns in the patient’s family. RNA sequencing (RNA-seq) and reverse-transcriptase–PCR analyses revealed missplicing of exon 6 into a cryptic splice-acceptor site (i6.SA) in MFSD8 intron 6, in a location 119 bp upstream from the SVA insertion site, in blood samples and lymphoblasts from the proband and mother (but not the father or an unaffected sibling). This missplicing precisely segregated with the SVA insertion (Figure 1C and Fig. S5) and was predicted to lead to premature translational termination, which supported the pathogenicity of the insertion.

Development of an Antisense Oligonucleotide Drug

Figure 2. Figure 2. Antisense Oligonucleotide Drug Development. Panel A shows the location and chemistry of the ASOs that were designed to block the i6.SA splice acceptor site or exonic splicing enhancer (ESE) elements. (Additional details are provided in Table S1.) The ESE elements were predicted with RESCUE-ESE and ESEfinder.14,15 2′-MOE denotes 2′-O-methoxyethyl, and 2′-OMe 2′-O-methyl. Panel B shows the ratio of the normal exon 6–exon 7 (E6–E7) splicing to the abnormal exon 6–intron 6 (E6–i6) splicing (normalized to a no-transfection control), measured in patient fibroblasts that were transfected (for 24 hours at 100 nmol per liter) as indicated. To measure splice isoform-specific levels, multiplex reverse-transcriptase polymerase chain reactions were conducted with isoform-specific primer sets, and then the intensity of the isoform-specific bands was quantified by gel electrophoresis (Fig. S6). “Scrambled” indicates a nontargeting oligonucleotide (TY772). 𝙸 bars indicate 95% confidence intervals of the means. P values were calculated by two-sided t-test. Panel C shows RNA sequencing (RNA-seq) analysis validation of the splice-correcting effect of milasen (TY777). For the calculation of the fraction of normal splicing (exon 6–exon 7), three other splicing events that are mutually exclusive with the normal splicing were considered. Splicing events supported by only one read are not shown. P values were calculated by Fisher’s exact test. Panel D shows intracellular vacuoles, visualized by electron microscopy, in control fibroblasts (MFSD8 wild-type human foreskin fibroblast; BJ cell line) and in patient fibroblasts that are either untreated or transfected with the indicated oligonucleotide. Scoring was performed on a scale of 0 to 5, with 0 representing the lowest and 5 representing the highest level of vacuole accumulation.

Nusinersen is an FDA-approved antisense oligonucleotide drug for spinal muscular atrophy8-10 that changes the splicing pattern of the SMN2 RNA.11-13 Reasoning that an antisense oligonucleotide might be similarly used to correct missplicing and restore MFSD8 expression in our patient, we designed antisense oligonucleotides to target the i6.SA cryptic splice-acceptor site and nearby splicing enhancers (Figure 2A). When we tested these antisense oligonucleotides in patient fibroblasts, we identified three that boosted normal:mutant splicing ratios by a factor of 2.5 to 3. TY777 was the most efficacious (Figure 2B) and became our lead candidate; we dubbed it “milasen.”

Milasen is a 22-nucleotide antisense oligonucleotide with the same backbone and sugar chemistry modifications (phosphorothioate and 2′-O-methoxyethyl) as nusinersen.8,13 Dose–response analysis indicated that its half-maximal potency was in the nanomolar range. RNA-seq from patient fibroblasts showed that milasen treatment more than tripled the amount of normal (exon 6–exon 7) splicing (Figure 2C). Computer-based sequence analysis of milasen showed little potential for off-target binding in the human genome.

Fibroblasts from the patient had several cellular phenotypes characteristic of lysosomal dysfunction, including intracellular vacuolation,16 increased total lysosomal mass,17 autofluorescence, miscompartmentalization of lysosomal enzyme activity, and reduced autophagic flux18 (Figure 2D). All these phenotypes were alleviated by milasen administration, indicating that treatment could rescue not only splicing but also function. (Additional details regarding the analyses we performed during the development of milasen are provided in Tables S1 and S2 and Figs. S6 through S12.)

Studies in Support of an Investigational New Drug Application

The patient’s condition continued to deteriorate. Clinical evaluation at 7 years of age revealed an inability to make discernible words, dysphagia (prompting gastrostomy tube placement), the need for substantial support to walk, and 15 to 30 overt seizures per day. She did, however, remain alert and reactive to familiar stimuli and responded happily to hearing her favorite books and songs.

In the largest published case series of CLN7 Batten’s disease, the mean age of patients at onset was 3.3 years, and seven patients died at a mean age of 11.5 years.19 Considering our patient’s clinical prognosis, and after appropriate scientific and ethics review, we filed for FDA permission to initiate clinical investigational treatment under an Expanded Access Investigational New Drug application. Milasen drug substance (18 g) was manufactured and formulated for clinical administration. To identify potential hazards, we administered milasen to rats by intrathecal injection at three doses: 0.06 mg, 0.25 mg, and 1.0 mg (approximately 2.5 times, 10 times, and 42 times, respectively, the typical clinical dose of nusinersen, after compartmental scaling considerations). No adverse effects were observed in the group that received 0.06 mg. At higher doses, some animals (half of those in the group that received 0.25 mg and most of those in the group that received 1.0 mg) had hindlimb weakness, which resolved by approximately 24 hours after each dose. (For additional details, including multidose experiments, see Fig. S13 and Supplementary Text.) These results provided translatable indicators of potential toxic effects that could be readily monitored and guided our clinical protocol design.

Clinical Study

Figure 3. Figure 3. N-of-1 Clinical Study. Panel A shows the dosing schedule. (Additional details are provided in Fig. S14A.) Panel B shows the concentration of milasen in cerebrospinal fluid (CSF) before each administration (trough). An additional measurement of the concentration in CSF was obtained at day 174 (without concurrent dose administration). 𝙸 bars indicate the minimum and maximum values of duplicate measurements. Trough levels rose steadily in a dose-proportional fashion until day 40, at which point they dropped to 1.7 ng per milliliter and then resumed their rise with repeated dosing up to a plateau of 18 to 27 ng per milliliter. The dip at day 40 may have been due to a CSF leak, given its coincident timing with a post–lumbar puncture headache after the previous dose. A similar plateauing of CSF trough levels was observed in a previous study of intrathecally delivered nusinersen (9 to 11 ng per milliliter after four repeated doses of 12 mg).8 Panel C shows the trends in seizure frequency and duration as reported in a seizure diary recorded by the parents. Seizures were all of the same type: sudden startle followed by uncontrollable, untriggered laughter that was different from the patient’s natural laugh, at times accompanied by an increase in the nonspecific repetitive hand movements she had at baseline. Panel D shows the trends in seizure activity as detected by electroencephalography (EEG). In a comparison of the means of the initial two recordings and the subsequent three recordings, the daily seizure count, seizure duration, and percent cumulative time spent in seizure decreased by 63% (from 31.5 to 11.7 per day), 52% (from 108 seconds to 52 seconds), and 85% (from 3.9% to 0.6%), respectively.

We initiated a clinical study of milasen in our patient 1 month after we started the toxicology studies in animals (Figure 1A). Our regimen was roughly modeled after nusinersen,8-10 because of the parallels between the two drugs (i.e., they are similarly sized antisense oligonucleotides with identical chemical modifications, targeting the same tissue [the CNS]). Milasen was given by intrathecal bolus injection, starting at 3.5 mg and increasing approximately every 2 weeks up to 42 mg (Figure 3A and Fig. S14A). A dose of 42 mg was chosen because antisense oligonucleotides administered intrathecally to humans or nonhuman primates distribute to the brain (the target of milasen) with approximately one third the efficiency with which they distribute to the spinal cord (the target of nusinersen),8,13 and 42 mg of milasen is the molar equivalent of 3 times the typical nusinersen dose (12 mg). After dose escalation, two additional loading doses were administered, followed by maintenance dosing approximately every 3 months.

Clinical Outcomes

Adverse Events, Pharmacokinetics, and Imaging

Through the first year of treatment, no serious adverse events occurred. No clinically significant adverse changes were observed in vital signs; in the results of physical examination, including assessments of strength, gait, and sensory testing; or in clinical laboratory test profiles. Pharmacokinetic analysis indicated a general trend of dose-proportional increases in drug levels in cerebrospinal fluid (Figure 3B) with only limited systemic exposure. MRI of the head showed continued brain volume loss 7 months after treatment initiation, extending a trend that had been observed over the previous 3 years. In a previous study of gene therapy for CLN5 Batten’s disease, animals treated after symptom onset continued to have brain volume loss over a period of 20 months, despite treatment slowing the progression of symptoms.20 (Details of the results of these analyses in our study are provided in Tables S3 through S5 and Figs. S14 through S18.)

Neurologic and Neuropsychological Assessments

Testing with the use of Vineland Adaptive Behavior Scales, Second Edition (Vineland-II), showed declines in 7 of 11 neurologic and neuropsychological subscores between day −100 and day −6. Of the 4 subscores that did not decline, 2 remained the same and 2 improved in the 3 months before the initiation of the clinical study. These declines represented ongoing losses of some of the patient’s few remaining adaptive skills in the domains of communication, daily living skills, and socialization. Subscores tended to stabilize in the period from the start of the clinical study to approximately 3 months (3 declined, 6 remained the same, and 2 improved from day −6 to day 107) and from 3 months to 6 months (4 declined, 2 remained the same, and 5 improved from day 107 to day 203). The results of testing with the Bayley Scales of Infant and Toddler Development, Third Edition; Global Motor Function Measure–88 (GMFM-88) scores; and results of sensory threshold testing remained stable throughout treatment. (Details of these results are provided in Tables S4 and S5 and Figs. S15 through S17.)

Seizures

At baseline, our patient was having approximately 15 to 30 seizures per day, each lasting 1 to 2 minutes (Figure 3C). Over the course of the clinical study, the frequency decreased to between 0 and 20 seizures per day, and the duration of each seizure decreased to less than 1 minute. These trends were corroborated by overnight electroencephalographic monitoring (Figure 3D), which indicated that both the frequency and duration of seizures decreased by greater than 50% (comparing the initial two recordings and the subsequent three recordings). The percent cumulative time spent in seizure decreased by greater than 80%. No changes were made to the patient’s antiepileptic drug regimen (topiramate, 72 mg twice a day), and serum drug levels of topiramate remained stable throughout treatment (10 to 12 μg per milliliter at days 13, 68, 209, and 301).