A Study of the Safety and Preliminary Efficacy of NKG001 in Pediatric Patients With Type 1 or Type 2 Spinal Muscular Atrophy

A Single-Center, Open-Label, Single-Arm, Non-Randomized, Single-Dose, Dose-Escalation Investigator-Initiated Trial to Evaluate the Safety, Tolerability, and Preliminary Efficacy of NKG001 Injection in Patients With Type 1 or Type 2 Spinal Muscular Atrophy

This study is a single-center, open-label, single-arm, non-randomized, single-dose, dose-escalation investigator-initiated trial (IIT) designed to evaluate the safety, tolerability, and preliminary efficacy of NKG001 Injection administered via different dosing regimens (intravenous [IV] alone or intravenous combined with intrathecal [IV+IT]) in subjects with Type 1 or Type 2 spinal muscular atrophy (SMA).

A total of 13-21 SMA subjects aged ≤60 months are planned to be enrolled. Based on age at enrollment, subjects will be stratified into two age cohorts for independent evaluation:

Age Cohort 1: subjects aged <24 months at dosing; Age Cohort 2: subjects aged ≥24 months and ≤60 months at dosing.

Eligible subjects must carry 2 or 3 copies of the SMN2 gene.

Note: Subjects with 3 SMN2 copies must be able to sit independently but unable to walk independently.

Four dose cohorts are planned as follows:

S1: 6.0 × 10^13 vg/kg, IV S2: 1.2 × 10^14 vg/kg, IV S3: 6.0 × 10^13 vg/kg, IV + 6 × 10^13 vg/person, IT S4: 6.0 × 10^13 vg/kg, IV + 1.2 × 10^14 vg/person, IT

Subjects in the S1 cohort (2 SMN2 copies and aged <24 months at dosing) and the S2 cohort (2 or 3 SMN2 copies and aged ≤60 months at dosing) will receive a single intravenous administration of NKG001 Injection.

Subjects in the S3 and S4 cohorts will receive a single administration of NKG001 Injection via combined intravenous and intrathecal routes. In each of these two cohorts, the first enrolled subject must have 2 SMN2 copies and be aged <24 months at dosing, while the remaining subjects may have either 2 or 3 SMN2 copies and be aged ≤60 months at dosing.

Inclusion Criteria

Subjects who meet all of the following criteria are eligible for enrollment in this study:

1. Subjects aged ≤60 months on the day of dosing, regardless of sex.

2. Subjects must have a genetically confirmed diagnosis of spinal muscular atrophy (SMA) caused by biallelic SMN1 mutations (deletion or point mutation). SMN2 copy number requirements are as follows:

   • S1 cohort and S2-A cohort: 2 copies of SMN2;
   • S2-B cohort: 3 copies of SMN2;
   • S3 and S4 cohorts: 2 or 3 copies of SMN2.

Notes:

1. Subjects in the S1 cohort and S2-A cohort may be enrolled regardless of the presence or absence of clinical symptoms.
2. Subjects with 3 copies of SMN2 must be able to sit independently but unable to walk independently. Independent sitting is defined according to the WHO Multicentre Growth Reference Study (WHO-MGRS) criteria as maintaining an upright seated position with the head erect for at least 10 seconds without support from the arms or hands.
3. The subject's legally authorized representative (LAR) must understand the purpose, potential risks, and rights associated with the study; agree to the subject's participation in all study procedures, assessments, and visits; and voluntarily sign the informed consent form (ICF).
4. During the study period, based on changes in the subject's clinical condition, the subject's LAR must be willing to comply with standard-of-care recommendations provided by the investigator, including nasogastric feeding, non-invasive mechanical ventilation, cough assist devices, and other supportive treatments as needed.

Exclusion Criteria

Subjects meeting any of the following criteria will be excluded from participation in the study:

1. Gestational age at birth <35 weeks (245 days).

2. During screening, oxygen saturation <96% while awake or asleep without supplemental oxygen or respiratory support.

3. During screening, subjects with moderate or greater swallowing impairment whose caregivers are unwilling to use alternative feeding methods to oral feeding.

4. Requirement for invasive ventilation or tracheostomy, or use of non-invasive ventilatory support for an average of ≥12 hours/day during screening.

5. Positive test results for any of the following: human immunodeficiency virus (HIV) antibody, hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) antibody, Treponema pallidum antibody, TORCH infection, or Epstein-Barr virus (EBV).

6. Presence of other severe infections or diseases that may pose unnecessary risk for gene replacement therapy, including but not limited to:

   1. Upper or lower respiratory tract infection requiring medical care or intervention (including systemic therapy, hospitalization, respiratory support, or supplemental oxygen) within 4 weeks prior to dosing, or severe non-pulmonary/non-respiratory infection;
   2. Known epilepsy;
   3. Diabetes mellitus;
   4. Idiopathic hypocalcemia;
   5. Severe cardiovascular or cerebrovascular disease;
   6. Severe hepatic or renal impairment.

7. Clinically significant abnormal laboratory findings, including:

   • GGT, ALT, total bilirubin, or AST >3.0 × upper limit of normal (ULN) (isolated bilirubin elevation attributable to physiologic neonatal jaundice is not exclusionary);
   • Serum creatinine above the normal range;
   • Hemoglobin <80 g/L or >180 g/L;
   • White blood cell count <4.3 × 10^9/L or >14.2 × 10^9/L;
   • Platelet count <183 × 10^9/L or >614 × 10^9/L;
   • Prothrombin time (PT) prolonged by >1.0-1.5 × ULN.

8. Anti-AAV9 binding antibody titer >1:50 (measured by ELISA) or anti-AAV9 neutralizing antibody titer >1:200 (cell-based assay). Subjects exceeding these thresholds may undergo one repeat test during screening; subjects meeting eligibility criteria upon retesting may continue screening.

9. Known hypersensitivity or allergic predisposition to prednisolone acetate, other glucocorticoids, or any excipients of the investigational product.

10. Previous or planned treatment with other SMA gene replacement therapies (e.g., Zolgensma) during the study period.

11. Participation in another clinical trial within 1 month prior to screening, or prior participation in a clinical study that, in the investigator's judgment, may affect subject safety or evaluation of the investigational product, even if completed >1 month previously.

12. Receipt of immunosuppressive therapy within 3 months prior to dosing, or anticipated receipt within 3 months after study initiation, other than protocol-required prophylactic medications. Examples include corticosteroids, cyclosporine, tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin (IVIG), and rituximab.

13. Current use of, or inability to discontinue during the study, immunomodulatory agents (e.g., thymosin, interferons), medications for myopathy, neuritis, or diabetes (e.g., immunosuppressants, glucocorticoids, insulin), or receipt of plasmapheresis.

14. Oral treatment with β2-adrenergic agonists within 30 days prior to dosing (except inhaled salbutamol/albuterol).

15. Anticipated need for major surgery during the treatment period.

16. Prior to dosing, incomplete or delayed vaccinations according to the national immunization schedule for the subject's age that, in the judgment of the investigator and sponsor, may significantly affect subject safety.

17. Any other condition that, in the investigator's opinion, makes the subject unsuitable for participation in the study.

    Additional Exclusion Criteria for S3 and S4 Cohorts

    Subjects meeting any of the following criteria will be excluded from enrollment into the S3 and S4 dose cohorts:

18. Presence of cerebrospinal fluid circulation disorders or related diseases.

19. Contraindications to lumbar puncture or intrathecal administration, including but not limited to infection at the injection site, signs or symptoms of increased intracranial pressure, implanted cerebrospinal fluid drainage devices, or implanted central nervous system catheters.

20. Severe scoliosis (Cobb angle ≥50°), severe joint contracture deformities that may interfere with motor function assessments, or planned spinal corrective surgery during the observation period.