nature medicine

Brief Communication https://doi.org/10.1038/s41591-024-03078-4

AAV gene therapy for hereditary spastic

paraplegia type 50: a phase 1 trial in a
single patient

Received: 2 August 2023 James J. Dowling 1,2,3 , Terry Pirovolakis4, Keshini Devakandan1, Ana Stosic1,2,
Mia Pidsadny1, Elisa Nigro2, Mustafa Sahin 5, Darius Ebrahimi-Fakhari5,
Accepted: 20 May 2024
Souad Messahel6, Ganapathy Varadarajan6, Benjamin M. Greenberg 6,
Published online: xx xx xxxx Xin Chen6, Berge A. Minassian6, Ronald Cohn 1,3, Carsten G. Bonnemann 7 &
Steven J. Gray 6
Check for updates

There are more than 10,000 individual rare diseases and most are without
therapy. Personalized genetic therapy represents one promising approach
for their treatment. We present a road map for individualized treatment of
an ultra-rare disease by establishing a gene replacement therapy developed
for a single patient with hereditary spastic paraplegia type 50 (SPG50).
Through a multicenter collaboration, an adeno-associated virus-based gene
therapy product carrying the AP4M1 gene was created and successfully
administered intrathecally to a 4-year-old patient within 3 years of diagnosis
as part of a single-patient phase 1 trial. Primary endpoints were safety and
tolerability, and secondary endpoints evaluated efficacy. At 12 months
after dosing, the therapy was well tolerated. No serious adverse events
were observed, with minor events, including transient neutropenia and
Clostridioides difficile gastroenteritis, experienced but resolved. Preliminary
efficacy measures suggest a stabilization of the disease course. Longer
follow-up is needed to confirm the safety and provide additional insights
on the efficacy of the therapy. Overall, this report supports the safety
of gene therapy for SPG50 and provides insights into precision therapy
development for rare diseases. Clinical trial registration: NCT06069687.

Rare diseases affect more than 400 million persons. They are associ- Hereditary spastic paraplegia type 50 (SPG50) is a prototypical
ated with considerable disabilities, early mortality and disproportion- ultra-rare (affecting <1 in 50,000) disease, with fewer than 100 affected
ate impacts on the healthcare system. Less than 5% have treatments, individuals identified3,4. It is caused by biallelic pathogenic variants in
highlighting a critical need for new therapies. There is now the concep- the AP4M1 gene, encoding a subunit of the adaptor protein complex
tual ability to develop gene- and/or mutation-specific treatments for 4 (AP-4)5–9. Symptom onset is typically in infancy and includes global
many rare diseases1,2. However, important barriers exist, particularly developmental delay, progressive microcephaly and abnormalities
related to patient numbers, development costs and lack of financial on brain magnetic resonance imaging (MRI)3,4,10. The disease is pro-
incentives. gressive, with loss of motor skills due to worsening spasticity, and is

1
Precision Child Health, Hospital for Sick Children, Toronto, Ontario, Canada. 2Division of Neurology and Program for Genetics and Genome Biology,
Hospital for Sick Children, Toronto, Ontario, Canada. 3Departments of Paediatrics and Molecular Genetics, University of Toronto, Toronto, Ontario,
Canada. 4CureSPG50 Foundation, Toronto, Ontario, Canada. 5Department of Neurology, Boston Children’s Hospital, Boston, MA, USA. 6Department of
Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA. 7Neuromuscular & Neurogenetic Diseases of Childhood, Neurogenetics
Branch (NGB), Bethesda, MD, USA. e-mail: [email protected]

Nature Medicine
Brief Communication https://doi.org/10.1038/s41591-024-03078-4

associated with serious morbidities3,11. By the second decade of life, Clostridioides difficile gastroenteritis and side effects of tacrolimus. We
most affected individuals are wheelchair dependent and manifest detected no clinical or electrophysiological evidence of dorsal root
severe cognitive dysfunction. Lifespan is not fully established, but the ganglion (DRG) toxicity; there were no neuropathic pain complaints,
disorder is considered life-limiting. and the results of sensory examination and nerve conduction studies
SPG50 is an ideal candidate disease for gene therapy. The coding were normal (Extended Data Fig. 6). Contrast-enhanced brain and
sequence is small (1,359 base pairs) and fits within a self-complementary spine MRI at 3, 6 and 12 months after dosing showed no inflammatory
adeno-associated virus (scAAV) vector. Causative mutations result in changes and no progression in brain atrophy.
loss of expression/function, so gene re-expression is anticipated to be Progressive limb spasticity is a major SPG50 disease component11.
effective, and the nature of the AP-4 complex as an obligate heterote- We measured spasticity using two scales previously developed for cer-
tramer may protect against overexpression-related toxicity12. There is ebral palsy: the Tardieu16 and modified Ashworth17 scales. These were
a relatively large therapeutic window, as disease progression occurs not well tolerated (due to the patient’s discomfort with passive joint
over years, with potential for functional benefit likely before irrevers- manipulation), and data points across several assessments are missing
ible disability. However, the disorder’s rarity precludes typical drug (Extended Data Figs. 7 and 8). However, compared to predosing assess-
development pathways. ments, there was no negative change in successfully scored joints.
We present a case wherein gene therapy was developed for a single Developmental delay is also an important feature of SPG50. We
male patient with SPG50 (Fig. 1a). The disease was diagnosed at age examined this using two exploratory measures: the Bayley Scale of
18 months by whole-exome sequencing (AP4M1 c.916 C>T, p.R306X; Infant and Toddler Development18 and the Vineland Adaptive Behav-
c.696dupG, p.E232GfsX21) based on a presentation of developmental ior Scale19. Bayley scores increased across multiple domains (Fig. 2b).
delay (unable to stand or walk independently, no word production) Vineland scores were more variable, with a modest decline in adaptive
and microcephaly. At diagnosis, based on our international registry behavior and improvements in motor domains (Fig. 2c and Extended
(NCT04712812), the proband was the only Canadian individual with Data Fig. 9).
SPG50. Shortly after diagnosis, the family created the CureSPG50 At the time of the last examination, the patient was able to stand
Foundation with the goal of developing SPG50 gene therapy. At the with his heels on the ground (Clinical Global Impression (CGI) of
predosing baseline, the patient could crawl 5 feet, pull himself up to Improvement (CGI-I) level 3 = minimally improved; Methods)—some-
stand momentarily at a table and walk a few steps with assistance. He thing that had not been achieved before dosing—and to subjectively
had a pincer grasp and could feed himself with his hands, stack two tolerate longer periods of standing in a stander and walking with an
blocks and scribble. He was nonverbal and had limited communication assist device. No subjective disease worsening or loss of skills was
with gestures and nonword sounds. Physical examination was most observed. The parent log data showed that, since receiving the therapy,
notable for diffuse spasticity (lower extremity more affected than the patient has not experienced falls or seizures. Before dosing, the
upper extremity) and hyperreflexia. patient had infrequent seizures (one seizure in the previous 24 months).
The investigational product was designed based on similar vectors Overall, we describe the full development cycle of a single-patient
made for CLN7 disease and giant axonal neuropathy13 and includes gene therapy for SPG50. Typically, the implementation of new treat-
codon-optimized human AP4M1 driven from the JeT promoter and ments comes too slowly to help the patient(s) that initially inspired
encapsulated into scAAV9 (AAV9-AP4M1; Extended Data Fig. 1)14. them. This study represents an example of AAV-based gene therapy
Based on preclinical data14, a safety, toxicity and efficacy package for that was rapidly developed and administered in a timely fashion to
AAV9-AP4M1 was filed to Health Canada, along with a clinical protocol benefit the original ‘inspirational’ patient. Therefore, it provides a
and information on chemistry, manufacturing and control. A ‘no objec- potential road map for individualized genetic therapy for other ultra-
tion letter’ was received in December 2021, 2 years and 8 months after rare disorders.
diagnosis. The study protocol enumerated the eligibility criteria and The primary outcome was safety, and no serious adverse events
safety assessments based on the gene therapy trial for giant axonal neu- were identified despite the large dose of AAV administered intrathe-
ropathy (NCT02362438)15, and efficacy measures were derived from cally. Our immunosuppression protocol was more extensive than
the ongoing SPG50 natural history study (NCT04712812). Institutional that used in many previous gene therapy trials, reflecting our concern
ethics board approval was obtained in February 2022. Although the about immune-mediated toxicities and our desire to promote lasting
trial was not registered with ClinicalTrials.gov until October 2023, all immune tolerance to the gene therapy product. While some observed
inclusion and exclusion criteria, safety studies and outcome measures side effects were attributable to immunosuppression, our patient also
were established before study initiation and patient enrollment. did not develop an anti-AP4M1 immune response. Determining whether
A single-patient trial (NCT06069687) was initiated (Fig. 1b), this represents an optimal immunomodulation strategy for AAV9 gene
with dosing in March 2022, 2 years and 11 months after diagnosis. therapy will require its use in additional patients and gene therapy
The primary outcome was safety, and secondary efficacy measures programs. Of note, our patient experienced transient neutropenia and
were related to spasticity. AAV9-AP4M1 was administered at 1 × 1015 a T cell reaction to AAV9 (Extended Data Fig. 2), suggesting that some
vector genomes (vg) through intrathecal delivery. This is among the AAV9 had entered the systemic circulation.
largest doses of AAV9-based gene therapy ever administered into the Regarding efficacy, our assessments indicated possible disease
cerebrospinal fluid (CSF). stabilization after AAV9-AP4M1 treatment. Based on existing natural
We used an extensive immunosuppression protocol (pred- history data, progression is anticipated over a 1-year period3. Thus,
nisolone, sirolimus and tacrolimus) designed to reduce adverse our data may represent a modification of the expected disease course.
immune responses and promote tolerance to the AP4M1 protein, A notable aspect of this study was its rapid development. The
given the patient’s predicted lack of endogenous expression. Based time from diagnosis to dosing was <3 years. The speed of develop-
on enzyme-linked immunospot (ELISpot) data, the patient has not ment was aided by several factors, including the use of an existing
developed any appreciable anti-AP4M1 response (Extended Data Fig. 2). AAV9-based gene therapy ‘template’ and collaboration between mul-
No serious adverse events were detected through 12 months after tiple researchers. This latter aspect was facilitated by the CureSPG50
dosing. Notable safety-related events are presented in Fig. 2a, with all Foundation, which nucleated the work and established connections
safety data listed in Extended Data Figs. 3–5. Neutropenia was noted 6 between researchers, clinicians, contract research organizations and
days after dosing, which resolved without intervention within 1 week. industry partners.
At 5 months after dosing, the patient experienced severe abdominal dis- There may be opportunities to accelerate future projects further.
comfort, which has since resolved and was ultimately attributed to both Preclinical SPG50 models had to be established. For other diseases,

Nature Medicine
Brief Communication https://doi.org/10.1038/s41591-024-03078-4

a
2019 2020 2021 2022

Initial First clinical Rat GLP Tox NHP GLP Tox

diagnosis description of study ends study completed Submission
SPG50 published to FDA
Approval to
proceed (Health
Proof of Toxicology Canada)
Founder
concept begins batch
mice FDA pre-IND Clinical
(UTSW) created Clinical batch
received response batch
created
shipped

2 Apr 9 May 12 Dec 9 Mar 12 Aug 1 Sep 26 Sep 10 Dec 22 Dec 9 Feb 12 May 14 May 23 May 22 Jun 6 Jul 13 Jul 14 Oct 30 Nov 1 Dec 30 Dec 23 Feb 24 Mar 10 Jul 11 Aug

12-Month AP4M1 5- Clinical

mouse Tox month mouse batch COA
study begins data released
12-Month
mouse Tox
International study ends
registry and natural Health Canada Submission to Gene therapy
history study pre-CTA meeting Health Canada administered
launched

NHP GLP Approval to

Manufacturing Rat GLP Tox proceed (FDA)
contract begins study begins Tox study
start

b Days Months

Timeline –7 –1 1 2 7 14 21 28 3 6 9 12

Transgene expression Adaptive → Tolerance → →

GT
Ongoing
Type 1

Type 1

Type 1
Type 1

Type 3

Type 3
Type 2

Type 2

Safety and
monitoring

Type 1: MRI, CSF, safety labs & exploratory tests Type 2: safety labs Type 3: safety labs & exploratory tests

Ongoing

Rapamycin maintenance
Pulse

IV
Immune MethylPred
protocol Prednisone maintenance Taper

Tacrolimus maintenance Taper

Fig. 1 | Development and implementation of individual gene therapy for ‘GT’ indicates the gene therapy dosing. MRI of the brain and spine (with and
SPG50. a, Timeline of the development of SPG50 gene therapy, from patient without contrast) was done at baseline and at 3, 6 and 12 months after dosing.
diagnosis through patient dosing, with key milestones highlighted. Note that CSF analysis included cell count, protein concentration, oligoclonal bands
the entire process, from diagnosis to dosing, took approximately 2.5 years. and cytokine analysis. Exploratory tests included measurement of the AAV9
UTSW, University of Texas Southwestern; FDA, Food and Drug Administration; neutralizing antibody titer, serum cytokine analysis and ELISpot assay. Safety
IND, investigational new drug; GLP, Good Laboratory Practice; NHP, nonhuman laboratory tests (‘safety labs’) included complete blood count with differential,
primate; Tox, toxicology; CTA, clinical trial application; COA, certificate of erythrocyte sedimentation rate, C-reactive protein, liver function tests (alanine
analysis. b, Outline of the single-patient clinical trial. The schematic depicts aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, alkaline
the postdosing safety and efficacy monitoring time points, along with the phosphatase), blood urea nitrogen/creatinine, urinalysis, electrocardiography
immunosuppression protocol. The comprehensive immunosuppression and cardiac safety panel (troponin, pro-B-type natriuretic peptide, creatine
program was implemented to attempt to minimize the innate and adaptive kinase isotype MB). IV MethylPred, intravenous methylprednisolone.
immune responses and to promote tolerance to the gene therapy product.

these could be developed in advance of therapy conception. Toxic- including DRG toxicity. This highlights a broader question of the pre-
ity experiments in nonhuman primates were strongly encouraged dictiveness of animal studies for safety and toxicity, something that
by regulatory agencies. As more gene therapy trials are successfully has come to light with other gene therapy programs, in which there
completed, the requirement for such studies may be reduced. None of has been safety signal discordance between animal toxicology studies
the preclinically identified adverse findings presented in our patient, and human clinical trials20,21.

Nature Medicine
Brief Communication https://doi.org/10.1038/s41591-024-03078-4

a
Duration
Onseta Resolutiona (days) Relationship to study drug

Serious adverse events

None – – – –
Adverse events related to IP
Neutropenia Day 6 Day 13 7 Probable
Adverse events related to other treatment
Vomiting (emesis)
Day 3 Day 3 1 None
Prednisone taste
Excessive fatigue Day 28 Day 58 30 Not likely
C-diff infection
Diarrhea, vomiting,
Day 177 Day 263 86 Not likely
dehydration
Tacrolimus side effect
Adverse events unrelated to IP
Upper respiratory tract
Day 35 Day 47 12 Not likely
infection
a
Days from IP administration.

b 90
Bayley subset raw score profile

80

70

60

50

40

30

20

10

0
Day –1 Month 3 Month 6 Month 9 Month 12

Cognitive Receptive communication Expressive communication

Fine motor Gross motor

c
Receptive Expressive
Study visit Cognitive Fine motor Gross motor
communication communication

Day –1 6 25 16 48 31
Month 3 6 12 15 36 4
Month 6 81 27 16 56 60
Month 9 77 20 16 54 63
Month 12 84 21 15 51 60

d
Month
Subdomains Screening Day –1 Month 3 Month 9 Month 12
6

Raw score – 17 23 20 23 22
Gross
motor
V-scale score – 2 3 2 3 3

Raw score 25 24 25 14 17 28
Motor Fine
skills motor
V-scale score 1 8 8 4 5 8

Sum of V-scale score – 10 11 6 8 11

Standard score – 48 57 36 42 57

Fig. 2 | Safety and efficacy (Bayley Scale of Infant Development) in the SPG50 b, Graphical representation of the longitudinal results of the Bayley Scale of
single-patient therapy trial. a, Enumeration of the adverse events reported Infant and Toddler Development, fourth edition. From 6 months after dosing,
in the clinical trial over the 1 year after dosing (IP, investigational product). there were consistent increases in scores for all domains except expressive
No serious adverse events were observed. The patient experienced transient, communication. This mirrors what was qualitatively observed by both the family
asymptomatic neutropenia noted at 6 days after dosing. This resolved without and the examination team. c, Presentation of the longitudinal raw data from
intervention by day 13 after dosing. There was a prolonged episode of abdominal the Bayley scale (visualized graphically in b). Of note, the baseline and 3-month
symptoms that included emesis, diarrhea, vomiting and abdominal pain. This studies were complicated by challenges with the patient’s tolerance of the test.
episode prompted extensive evaluation, with the ultimate conclusion that the d, Scores from the motor skills submodule of the Vineland Adaptive Behavior
symptoms were due to side effects of tacrolimus plus C. difficile (C-diff) infection. Scale. Improvements were noted in both fine and gross motor performance.

Nature Medicine
Brief Communication https://doi.org/10.1038/s41591-024-03078-4

The trial design was innovative although not unique, as other time that may be unnecessary in settings like this (ultra-rare disease,
single-patient genetic therapy trials have been completed22. We used high unmet need, use of an existing vector backbone).
emerging data on the disease’s natural history combined with the In conclusion, we present an individualized gene therapy trial and
patient’s pretreatment data to monitor and assess efficacy—a strategy outline a path for future similar studies for ultra-rare diseases. Subse-
potentially applicable to future studies. Spasticity was a challenging quently, this study has motivated a larger United States-based trial to
outcome to measure, particularly in this young, nonverbal patient treat additional patients with SPG50 (NCT05518188).
who did not tolerate extensive direct examination. Therefore, existing
scales may not be suitable for some patients with spastic paraplegia. Online content
One future outcome could be timed heel versus toe standing, as this Any methods, additional references, Nature Portfolio reporting sum-
reflects ankle spasticity and range of motion and has functional links maries, source data, extended data, supplementary information,
with pathologic toe walking. acknowledgements, peer review information; details of author contri-
More generally, for single-patient trials, it is crucial to establish butions and competing interests; and statements of data and code avail-
objective and easily measurable outcomes. In individuals with epilepsy ability are available at https://doi.org/10.1038/s41591-024-03078-4.
or abnormal involuntary movements, quantification of seizures or
movements can provide a robust measure of treatment response. References
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ambulant individuals. Early-phase studies can thus serve important oligonucleotide therapy. Nature https://doi.org/10.1038/s41586-
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lenge with existing spasticity scales and identified a new possible for developing treatments for inherited disorders. Am. J. Hum.
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Methods to hematologic, immune and liver function and/or injury, as well as

Regulatory information and trial oversight assessment of DRG toxicity by nerve conduction studies. The secondary
Approval to proceed (that is, a no objection letter) was obtained from objective was to assess efficacy, which was determined by examining
Health Canada on 30 December 2021. The protocol (version 5) and sup- the patient for stability or improvement in spasticity (as assessed using
porting documentation were submitted to the SickKids Research Ethics the modified Ashworth and Tardieu scales).
Board (REB) on 7 January 2022. REB approval (REB no. 1000079110) Exploratory assessments included measurement of AAV9 antibody
was obtained on 15 February 2022. Protocol version 5 established the titers, evaluation of T cell responses to AAV9 and A4PM1 by whole-blood
inclusion/exclusion criteria and prespecified all safety and efficacy ELISpot assay, evaluations based on rating scales (Vineland Adaptive
outcome measures. Recruitment for the trial was opened at the time of Behavior Scale (Comprehensive Parent/Caregiver Form), CGI of Overall
the approval of protocol version 5. Subsequent amendments (versions Change by Physician, Bayley Scale of Infant and Toddler Development
5.1 and 6) to this protocol addressed minor changes to the immunosup- (fourth edition)), and use of logbooks to record the number and dura-
pression regimen, minor clarifications to the Bayley scale (removal of tion of seizures and falls daily.
the Growth Scale Value score) and reporting change for the Vineland The CGI assesses changes from the pretreatment baseline (CGI-I)
scale (switch from examiner to caregiver reporting). and the severity of the current illness (CGI-S). CGI-I is a seven-point scale
Before submission to Health Canada, a review of the proposed (1 = very much improved, 2 = much improved, 3 = minimally improved,
study was completed by an internal ethics committee. This committee 4 = no change, 5 = minimally worse, 6 = much worse and 7 = very much
included the chair of the REB, an expert bioethicist, in-house legal coun- worst). CGI-S is also a seven-point scale (1 = normal (shows no signs of
sel and members of the hospital executive leadership. The committee illness), 2 = borderline ill, 3 = slightly ill, 4 = moderately ill, 5 = markedly
discussed the challenges posed by this single-patient study, including ill, 6 = severely ill and 7 = among the most extremely ill of patients).
issues related to conflicts of interest and informed consent. Study sub-
mission proceeded after committee evaluation and incorporation of Inclusion and exclusion criteria
guidance related to trial elements, including consent and monitoring. Inclusion criteria.
Informed consent was obtained following the standard operat- • Age <5 years
ing procedure set by SickKids. Capacity assessment of the participant • Confirmed diagnosis of SPG50 disease by
was completed by the study doctor. Appropriately delegated research (a) Genomic DNA mutation analysis demonstrating homozy-
study team members discussed the informed consent statement with gous or compound heterozygous, pathogenic and/or likely
both parents. The study doctor was not present during the signing pathogenic variants in the AP4M1 gene
of the consent form (to avoid undue influence) but was available for (b) Clinical history or physical examination consistent with
discussion and clarification. Ample time was provided for the family SPG50
to ask questions and consider the trial. Upon discussion, the consent • Parent/legal guardian willing to provide written informed con-
form was signed by the delegated study coordinator and a parent on sent for their child before study participation
11 March 2022. The capacity to consent is assessed by the study doc- • Patient able to comply with all protocol requirements and
tor on an ongoing basis. If and when applicable, appropriate assent or procedures
consent will be obtained from the study participant.
Throughout the study, study conduct and data were monitored by Exclusion criteria.
the Clinical Research Quality and Education Board at SickKids.
Per Health Canada specifications, registration of trials in a pub- • Inability of the patient to participate in study procedures, as
lic database is encouraged. Owing to our uncertainty at the time of determined by the site investigator
obtaining the no objection letter regarding single-patient studies, the • Presence of a concomitant medical condition that precludes
study was initially not registered. It was retrospectively registered at lumbar puncture (LP) or use of anesthetics
ClinicalTrials.gov in October 2023 (NCT06069687). • History of a bleeding disorder or any other medical condition or
circumstance in which LP is contraindicated according to local
Vector design, manufacturing and dosing institutional policy
The design of AAV9-AP4M1 has been described previously14. The vector • Inability of the patient to be safely sedated, in the opinion of the
structure and sequence are presented in Extended Data Fig. 10. The clinical anesthesiologist
clinical AAV9-AP4M1 vector (MELPIDA) was manufactured by Viralgen • Active infection at the time of dosing, based on clinical
in accordance with current Good Manufacturing Practice standards. observations
Briefly, it was manufactured using Viralgen’s proprietary process • Concomitant illness or requirement for chronic drug treatment
involving triple-plasmid transfection into suspension HEK293 cells, that, in the opinion of the principal investigator, creates unnec-
followed by downstream processing to remove impurities and enrich essary risks for gene transfer
for genome-containing AAV particles. The final solution of AAV9-AP4M1 • Inability of the patient to undergo MRI according to local institu-
was formulated in PBS containing 5% d-sorbitol and 0.001% pluronic tional policy
F68. The final certificate of analysis is provided as Supplementary • Inability of the patient to undergo any other procedure required
Data. A total dose of 1 × 1015 vg was delivered to the patient over 10 min in this study
at a volume of 10 ml by lumbar intrathecal administration with the • Presence of non-SPG50-related CNS impairment or behavioral
patient in 15° Trendelenburg positioning (head down). The patient disturbances that would confound the scientific rigor or the
was maintained in the Trendelenburg position for 1 h after infusion. interpretation of the study results
The dose was derived from preclinical studies and extrapolated from • Received an investigational drug within 30 days before screen-
calculations of normative CSF volumes. ing or plan to receive an investigational drug (other than gene
therapy) during the study
Study objectives • Enrollment and participation in another interventional
The primary objective of this study was to evaluate the safety and clinical trial
tolerability of a single dose of AAV-AP4M1 (that is, MELPIDA) adminis- • Contraindication to AAV-AP4M1 or any of its ingredients
tered intrathecally to a single child with SPG50. Safety was evaluated • Contraindication to any of the immunosuppressive medications
as described below; the evaluation included serum studies related used in this study

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Brief Communication https://doi.org/10.1038/s41591-024-03078-4

• Clinically significant abnormal laboratory values (γ-glutamyl per day divided two times a day. Levels were checked after
transferase (GGT), alanine aminotransferase (ALT) and aspartate 5 days of treatment and deemed to be within the acceptable
aminotransferase (AST) or total bilirubin more than three times range; thus, the therapy was continued at this dose. Prednisone
the upper limit of normal, creatinine ≥1.5 mg dl−1, hemoglobin (1 mg kg−1 per day) and tacrolimus (0.2 mg kg−1 per day divided
<6 or >20 g dl−1, white blood cell count >20,000 per mm3) two times a day) were started 1 day after infusion. The levels
before therapy of both tacrolimus and sirolimus were monitored monthly.
At 3 months, prednisone taper was started, with completion in
Study procedure. 4 weeks. At 6 months, tacrolimus taper was initiated, with
completion in 4 weeks. Both tapers were initiated after a review
• Study initiation. A potential participant was identified. The of brain MRI and CSF analysis results confirmed no concern for
study team presented the study to the participant’s parents, active infection or inflammation. Sirolimus wean is planned to
and forms were given to the family for review. Time was start at 18 months.
provided for questions and study review. After discussion and • Postdosing assessments. At 7, 14, 21 and 28 days after infusion,
consideration, the delegated study coordinator obtained verbal the participant was brought on-site for a review of vital signs,
and written informed consent from the participant’s parents on safety laboratory tests, brief physical examination, collection of
11 March 2022. viral shedding samples, documentation of concomitant medica-
• Screening visit. A ‘screening visit’ was conducted. The screening tions and enumeration of any adverse events. On days 7 and 21,
visit (−28 to −8 days before vector infusion) included confirma- exploratory laboratory tests were performed. In addition, on
tion of the genetic diagnosis, review of medical history and day 21, nerve conduction studies were performed, and on day 28
concomitant medications, a complete physical examination, a comprehensive neurological physical examination was com-
vital sign assessment, height and weight measurements, pleted. At 3, 6, 9 and 12 months, the participant was assessed
15-lead electrocardiography, liver ultrasonography, blood and for all outcome measures. In addition, as a safety measure to
urine collections for safety laboratory tests, and spasticity monitor for CNS inflammation or infection, brain and spine MRI
assessments (modified Ashworth and Tardieu scales) performed (with and without gadolinium) and an LP for CSF analysis were
by a trained examiner. performed at baseline, 3, 6 and 12 months. For all LPs, a 21-gauge
• Safety laboratory tests. These tests included complete blood standard LP needle was used. EMLA (a eutectic mixture of local
count with differential, coagulation tests (international normal- anesthetics) was applied for local anesthesia, and then the LP
ized ratio, prothrombin time, partial thromboplastin time), needle was inserted into the intrathecal space between L4/L5.
erythrocyte sedimentation rate, C-reactive protein, Na, K, Cl, Ca, An appropriate quantity of CSF was removed for relevant safety
CO2, blood urea nitrogen, creatinine, glucose, ALT, AST, total/ laboratory studies (complete blood cell count with differential,
direct/indirect bilirubin, alkaline phosphatase, GGT, serum total protein, glucose, bacterial culture). Liver ultrasonography was
protein, cardiac safety panel (troponin, pro-B-type natriuretic conducted at 6 and 12 months. Nerve conduction studies were
peptide, creatine kinase isotype MB) and urinalysis (for protein, performed at 3, 6 and 12 months. Nerve conduction studies and
cells, glucose and bacteria). Laboratory samples were drawn brain MRI are planned at 18 months and 2 years after dosing and
at −28, −7 and −1 days before dosing and 2 days, 7 days, 14 days, then yearly thereafter. An additional LP will be performed at 18
21 days, 28 days, 3 months, 6 months, 9 months and 12 months months, before the planned sirolimus wean.
after dosing. • Documentation. Adverse events and concomitant medications
• Dosing. Dosing was accomplished through infusion into the were monitored on a continuous basis over the course of enroll-
intrathecal space. LP was performed through interventional ment and reviewed at each study visit. Any adverse events were
radiology with an anesthesiologist present to administer reported and documented in a timely manner and in accord-
sedation before infusion. The participant was placed in the ance with the regulatory requirements of SickKids and Health
Trendelenburg position (head down). An atraumatic Sprotte Canada. Data collection began at the time of informed consent
needle (Pajunk, item no. 321151-31A) was inserted percutane- signing. Source data included all information, original records
ously at the lumbar level L4/L5 interspace. Needle placement of clinical findings, observations and all clinical trial activities,
was confirmed with fluoroscopic intraoperative imaging before as necessary for the reconstruction and evaluation of the trial.
and after administration. Before infusion, 10 ml of CSF was Electronic case report forms were used to collect and store all
withdrawn from the lumbar space. MELPIDA solution was loaded study data in addition to maintenance of the original source
into a 20-ml BD syringe connected to the needle with 60-inch documentation. The electronic data capture platform used was
mini-volume intravenous extension tubing and a Braun four-way REDCap. Interim analyses were performed at 6 and 12 months
stopcock. The infusion was administered at a rate of 1 ml min−1, after dosing and are planned for yearly thereafter up to 5 years.
for a total of 10 ml, using a CareFusion Alaris 8110 syringe pump. As presented in section 9.1 of the protocol (‘Database locks’ in
Following administration, the participant remained in the Supplementary Information), interim analyses were prespeci-
Trendelenburg position (head down) for 1 h with turning (left to fied to be performed at periodic intervals per the judgment of
right, right to left) every 15 min. In addition, vital signs, including the study team (to review ‘key deliverables requiring analysis’).
heart rate, respiratory rate, blood pressure and pulse oximetry,
were monitored continuously for 1 h and then every 15 min until
2 h after infusion, every 30 min for the following 2 h (third and Sex and gender as biologic variables
fourth hour following infusion), hourly for an additional 4 h Given that this was a single-patient study (one male participant), we
and subsequently every 4 h until discharge. The patient was dis- are not able to adequately study or make conclusions regarding the
charged without complications on the day following MELPIDA potential impact of sex and/or gender on SPG50 and AAV9-AP4M1
administration. gene therapy.
• Immunosuppression. Three immunosuppressive agents were
used (sirolimus, tacrolimus and prednisone). Sirolimus was initi- Reporting summary
ated 1 week before infusion, with an initial load of 1 mg m−2 every Further information on research design is available in the Nature
4 h for three doses, followed by daily enteral dosing at 1 mg m−2 Portfolio Reporting Summary linked to this article.

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Brief Communication https://doi.org/10.1038/s41591-024-03078-4

Data availability for Health Canada. J.J.D., K.D., A.S., M.P. and E.N. executed the clinical
All data relevant to supporting the findings reported in this study are trial. J.J.D., K.D., A.S., D.E.-F. and S.J.G. analyzed the clinical data. J.J.D.
available within the paper and in the Supplementary Information. wrote the initial draft of the manuscript. J.J.D., T.P., K.D., A.S., M.P., E.N.,
Restrictions apply to some information related to the study, which are D.E.-F. and S.J.G. provided manuscript edits.
protected per institutional review board requirements. The sequence
and structure of MELPIDA are included as Extended Data Fig. 10. For Competing interests
all data inquiries, please contact Ana Stosic ([email protected]) S.J.G. and X.C. are inventors on a patent application for the AP4M1
and/or James Dowling ( [email protected]). Data or material vector design. Of note, T.P. is a parent of the study patient. Also,
transfer agreements may be required and will be assessed at the time subsequent to the completion of this study, T.P. formed Elpida
of request (approximate timeline for review = 8 weeks). Therapeutics, and MELPIDA (AAV-AP4M1) represents one of the clinical
programs in its developmental pipeline. S.J.G. is a nonpaid member of
Acknowledgements the Elpida board of directors, and S.M. is head of clinical operations.
We acknowledge the work of the SickKids clinical research unit and The other authors declare no competing interests.
supporting clinical services. In particular, thanks go to M. Shroff,
S. Miller, H. Gonorazky, F. Paiz and M. Bedford. This study was funded Additional information
by the sponsor (SickKids) through philanthropic donations to the Extended data is available for this paper at
SickKids Foundation and supported by the Precision Child Health https://doi.org/10.1038/s41591-024-03078-4.
initiative at SickKids. All funds went to support the trial, and the
individual authors received no specific funding for this work. We Supplementary information The online version
further acknowledge L. Black and D. Balderson for regulatory advice contains supplementary material available at
and support, as well as the Columbus Children’s Foundation for its role https://doi.org/10.1038/s41591-024-03078-4.
in providing the Good Manufacturing Practice (GMP) drug product.
The GMP/clinical-grade viral vector lot G-Geminis-029 was produced Correspondence and requests for materials should be addressed to
by Viralgen (San Sebastian, Spain). Funding from the CureSPG50 James J. Dowling.
Foundation supported the GMP drug product manufacture and
clinical trial application preparation. Peer review information Nature Medicine thanks Han-Xiang Deng,
Jonathan Kimmelman, Olivia Kim McManus and Terence Flotte for
Author contributions their contribution to the peer review of this work. Primary Handling
T.P., X.C., B.A.M. and S.J.G. conceived and developed the Editor: Anna Maria Ranzoni, in collaboration with the Nature Medicine
investigational product. M.S. and D.E.-F. defined the natural history team.
of the disease. J.J.D., T.P., D.E.-F., S.M., G.V., B.M.G., B.A.M., R.C., C.B.
and S.J.G. conceived the clinical implementation plan, developed the Reprints and permissions information is available at
clinical trial protocol and helped generate the regulatory submission www.nature.com/reprints.

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Brief Communication https://doi.org/10.1038/s41591-024-03078-4

Extended Data Fig. 1 | Schematic of the investigational product. Human, codon optimized AP4M1 (hAP4M1opt) with a bGH poly A tail was encapsulated into
self-complementary (sc) AAV9. AP4M1 expression was governed by a ubiquitous promoter (UsP = JeT promoter with intron). See Chen et al., 2023, Journal of Clinical
Investigation.

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Extended Data Fig. 2 | ELISpot assay reveals lack of immune response to the assay. Multiple two-tailed t-tests were conducted to assess for significant
AP4M1. (a) ELISpot to show IFN-y T-cell Responses toward AAV9. As expected, differences between treatment responses and negative controls. *P-values were
there is clear evidence of an immune response against AAV9. (b) ELISpot to show adjusted for multiple comparisons using the two-stage linear step-up method of
IFN-y T-cell Responses toward AP4M1. No significant response against AP4M1 Benjamini, Krieger, and Yekutieli (FDR = 5%).
was identified. (c) Positive control ELISpot used to confirm that the success of

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Brief Communication https://doi.org/10.1038/s41591-024-03078-4

Extended Data Fig. 3 | Safety lab trends during the 12 months post dosing. 7 post- dosing (0.8×109/L). ALT and GGT values were consistently outside of the
(a-d) Presented are the main safety laboratory studies, AST (a), ALT (b), GGT (c), normal range, but never reached a clinically meaningful increased level, and
and neutrophils (d), from baseline to 12 months post- dosing. Normative values remained < 2-fold above normal limits.
for age are highlighted in gray. There was a single instance of neutropenia at day

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Brief Communication https://doi.org/10.1038/s41591-024-03078-4

Extended Data Fig. 4 | Listing of laboratory studies performed during the study. Laboratory values obtained are listed from baseline through the first 12 months of
the study. Abnormal values (i.e. values outside the normal range) are highlighted in bold. Normative values, when available, are listed in the left most column.

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Brief Communication https://doi.org/10.1038/s41591-024-03078-4

Extended Data Fig. 5 | Listing of laboratory studies performed during the study (continued). Laboratory values obtained are listed from baseline through the
first 12 months of the study. Abnormal values (i.e. values outside the normal range) are highlighted in bold. Normative values, when available, are listed in the left
most column.

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Extended Data Fig. 6 | Sensory nerve analyses performed during the study. points. Intriguingly, amplitudes increased post-dosing, suggesting, if anything,
Standard nerve conduction studies were performed at baseline and then at improvements in sensory nerve function. Of note, NCS was also performed on
3 weeks, 3 months, 6 months, and 12 months. Presented are the data for the 5 the Tibial motor nerve, and all values were within normal limits (data not shown).
sensory nerves that were studied. Values were within the normal range at all time

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Brief Communication https://doi.org/10.1038/s41591-024-03078-4

Extended Data Fig. 7 | Scores of the Tardieu and modified Ashworth scales points. Tardieu scale values are 0 = no resistance to passive movement, 1 =
for the upper limbs. Scores from two measures of joint spasticity, the Tardieu slight resistance, 2 = clear ‘catch’, interrupting passive movement, 3 = clonus
and modified Ashworth scales. Existing natural history suggests worsening of ( < 10 seconds), 4 = sustained clonus. Ashworth scale values are 0 = no increase in
spasticity in SPG50 over a 12-month period. We observed stabilization of scores tone, 1 = slight increase in tone, 1 + = slight increase in tone, catch/release through
on both scales, with no clear worsening. However, the patient poorly tolerated range of motion, 2 = marked increase in tone, 3 = marked increase in tone AND
both outcome measures, resulting in missing data points at essentially all time passive movement difficult, 4 = fixed contracture.

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Brief Communication https://doi.org/10.1038/s41591-024-03078-4

Extended Data Fig. 8 | Scores of the Tardieu and modified Ashworth scales for the lower limbs. Scores for the lower limbs for the Tardieu and Ashworth scales.
Score values are presented in Extended Data Figure 7.

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Brief Communication https://doi.org/10.1038/s41591-024-03078-4

Extended Data Fig. 9 | Vineland Adaptive Behavior Scale (version 2). Results in adaptive behavior. This may be related in part to non-Melpida related side
from the Vineland adaptive behavior scale, parent reported. Substantial effects, including prolonged gastroenteritis and abdominal pain secondary to
gains were noted in both gross and fine motor skills (from baseline of 48 tacrolimus, as well as social impacts of immune suppression (such as prolonged
composite to 57 at 12 months post dosing). Small declines in scores were noted absence from school).

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Brief Communication https://doi.org/10.1038/s41591-024-03078-4

Extended Data Fig. 10 | AP4M1 vector structure and sequence. Melpida consists of a vector containing codon optimized AP4M1 and surrounding sequences
(UsP promoter and bGH polyA tail) inserted between truncated AAV2 ITR sequences and encapsulated in AAV9. (a) Schematic of the vector structure. (b) Sequence of
the vector.

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