Rajagopalan 2007.

Methods	Study design: multi‐centre double‐blind placebo‐controlled; 2 trial phases: first phase conducted as an RCT with n = 28 participants; second open‐label phase with n = 10 participants added in the treatment group and n = 3 original placebo‐treated participants rolled over to receive gene therapy Intention‐to‐treat: no, all participants receiving ≥ 1 HIF‐1α or placebo injection were included in the safety analysis Country: USA
Participants	Number randomised: N = 38 (HIF‐1α n = 31; placebo n = 7) Losses to follow‐up and withdrawals: not reported Age (mean years (range)): 66 (39 to 87) (HIF‐1α 66 (39 to 87); placebo 67 (46 to 80)) Gender (M): 66% (HIF‐1α 62%; placebo 100%) Inclusion criteria: between 21 and 45 years of age; no options for surgical or endovascular revascularisation and total or subtotal occlusion of at least 1 main artery in a limb confirmed by angiography; CLI (defined as Rutherford Category 4 or 5 present for a minimum of 4 weeks without response to conventional therapies with lack of further revascularisation options confirmed by both the investigator and an independent reviewer) Exclusion criteria: contraindications to growth factor therapy that have been published previously; inflammatory arthritis; Rutherford Category 6 status; prior successful lower extremity arterial surgery, angioplasty, or lumbar sympathectomy during the 2 months before screening; participated in other experimental protocols within 30 days of enrolment or had ever been enrolled in a similar vascular endothelial growth factor or fibroblast growth factor adenoviral or plasmid gene therapy protocol
Interventions	Treatment: Ad2/HIF‐1α /VP16 ‐ 1 × 10⁸ to 1 × 10¹⁰ viral particles (5 different treatment groups), 10 × 100 µL intramuscular injections for a total volume of 1.0 mL, into a single limb, placement of injections at discretion of investigator based on patient anatomy and location of occluded artery or arteries Control: placebo, phosphate‐buffered saline with 10% sucrose, given in the same manner as treatment
Outcomes	Follow‐up times: days 3, 7, 14, 21, 30, 45, 60, and 90, 6 months, and 1 year Outcomes: Adverse events, Changes in baseline physical examinations, Clinical laboratory evaluations, Adenoviral antibody titre measurement, Retinal eye examinations and examinations to assess rest pain, Healing of ischaemic ulcers, Rutherford Category, ABI, MRA to detect vascular changes
Notes	Study period: October 1999 to June 2004 Study reported pooled HIF‐1α results and not per dosage; we are reporting HIF‐1α as a single treatment group Treatment numbers reported in this review (HIF‐1α n = 31) differ from the report, as their n = 34 treated includes 3 participants originally randomised to placebo who were rolled over, so are counted twice (in the control group as well)
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	Insufficient information provided to determine random sequence generation
Allocation concealment (selection bias)	Unclear risk	Insufficient information provided to determine allocation concealment
Blinding of participants and personnel (performance bias) All outcomes	Unclear risk	First part of study was double‐blind, but second phase was open‐label, where several participants originally assigned to placebo were rolled over to treatment; blinding methods not described
Blinding of outcome assessment (detection bias) All outcomes	Unclear risk	Insufficient information provided to determine blinding of outcome assessment
Incomplete outcome data (attrition bias) All outcomes	Unclear risk	Information provided only as total trial, not separately by trial phases; withdrawals reported in Figures 2 and 3 but only for those with rest pain or ulcers
Selective reporting (reporting bias)	Low risk	All outcomes reported but ABI reportedly not available for all study participants
Other bias	High risk	Study sponsored by Genzyme Corp., manufacturer of Ad2/HIF‐1α/VP16 Study incorporated a randomised, double‐blind, placebo‐controlled first phase of the study with an open‐label phase after, where several placebo participants were rolled over to treatment and therefore were counted twice in the analysis