Abstract: We included in this review a total of 17 studies with 1988 participants (evidence current until November 2017). Three studies limited their inclusion to people with intermittent claudication, 12 limited inclusion to people with varying levels of critical limb ischaemia, and two included people with either condition. Study investigators evaluated many different types of gene therapies, using different protocols. Most studies evaluated growth factor-encoding gene therapy, with six studies using vascular endothelial growth factor (VEGF)-encoding genes, four using hepatocyte growth factor (HGF)-encoding genes, and three using fibroblast growth factor (FGF)-encoded genes. Two studies evaluated hypoxia-inducible factor 1-alpha (HIF-1α) gene therapy, one study used a developmental endothelial locus-1 gene therapy, and the final study evaluated a stromal cell-derived factor-1 (SDF-1) gene therapy. Most studies reported outcomes after 12 months of follow-up, but follow-up ranged from three months to two years. Overall risk of bias varied between studies, with many studies not providing sufficient detail for adequate determination of low risk of bias for many domains. Two studies did not utilise a placebo control, leading to risk of performance bias. Several studies reported in previous protocols or in their Methods sections that they would report on certain outcomes for which no data were then reported, increasing risk of reporting bias. All included studies reported sponsorships from corporate entities that led to unclear risk of other bias. The overall quality of evidence ranged from moderate to very low, generally as the result of heterogeneity and imprecision, with few or no studies reporting on outcomes. Evidence suggests no clear differences for the outcomes of amputation-free survival, major amputation, and all-cause mortality between those treated with gene therapy and those not receiving this treatment (all moderate-quality evidence). Low-quality evidence suggests improvement in complete ulcer healing with gene therapy (odds ratio (OR) 2.16, 95% confidence interval (CI) 1.02 to 4.59; P = 0.04). We could not combine data on quality of life and can draw no conclusions at this time regarding this outcome (very low-quality evidence). We included one study in the meta-analysis for ankle brachial index, which showed no clear differences between treatments, but we can draw no overall association (low-quality evidence). We combined in a meta-analysis pain symptom scores as assessed by visual analogue scales from two studies and found no clear differences between treatment groups (very low-quality evidence). We carried out extensive subgroup analyses by PAD classification, dosage schedule, vector type, and gene used but identified no substantial differences. Moderate-quality evidence shows no clear differences in amputation-free survival, major amputation, and all-cause mortality between those treated with gene therapy and those not receiving gene therapy. Some evidence suggests that gene therapy may lead to improved complete ulcer healing, but this outcome needs to be explored with improved reporting of the measure, such as decreased ulcer area in cm², and better description of ulcer types and healing. Further standardised data that are amenable to meta-analysis are needed to evaluate other outcomes such as quality of life, ankle brachial index, symptom scores, and claudication distance.

Plain-language summary: We included 17 studies that had a total of 1988 participants (evidence current until November 2017). These studies used various types of gene therapy as well as different dosages, some providing single treatments and some repeated treatments. Most of the studies included people with critical limb ischaemia; three studies included people with intermittent claudication. When combining the data, we found no clear differences between people who received gene therapy and those who did not in terms of amputation-free survival (patients who did not have an amputation and did not die), major amputation (above the ankle), or death. We did see improvement in complete ulcer healing in the gene therapy treatment group compared to the control group. Studies show no clear differences in pain symptom scores, but we evaluated only two studies for this outcome. Not enough data are available to show if there was a difference between groups for the measure of blood flow known as the 'ankle brachial index'. We were not able to combine data on quality of life or pain-free walking distances (distances one can walk without experiencing leg pain). Risk of bias of the included studies varied greatly, and this was a concern because studies did not clearly report on their methods nor on follow-up of participants. Most studies used a placebo control, which increases the risk that outcomes may have been different if people knew they were given treatment or control. Corporations that produce the tested treatments sponsored all included trials. The quality of evidence varied from moderate to very low. For amputation-free survival, major amputation, and death, we considered the quality of evidence to be moderate because of differences between studies. For ulcer healing, risk of bias was a matter of concern, and study results were imprecise because few events were reported. The quality of evidence for quality of life was very low because of differences between studies and insufficient information to combine study findings. The quality of evidence for the ankle brachial index was low because only one study with few participants reported this outcome. For pain symptom scores, the quality of evidence was very low because of technical problems within one of the two studies, as well as differences between the two studies and few participants.