pCMVvegf165 Intramuscular Gene Transfer is an Effective Method of Treatment for Patients With Chronic Lower Limb Ischemia

Original Manuscript

pCMV-vegf165 Intramuscular Gene Transfer
is an Effective Method of Treatment for
Patients With Chronic Lower Limb Ischemia

Journal of Cardiovascular
Pharmacology and Therapeutics
1-10
ª The Author(s) 2015
Reprints and permission:
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DOI: 10.1177/1074248415574336
cpt.sagepub.com

Roman V. Deev, MD, PhD1,2, Ilia Y. Bozo, MD1,3,4, Nina D. Mzhavanadze, MD, PhD5,
Dmitriy A. Voronov, MD, PhD6, Aleksandr V. Gavrilenko, MD, PhD, ScD6,
Yuriy V. Chervyakov, MD, PhD, ScD7, Ilia N. Staroverov, MD, PhD7,
Roman E. Kalinin, MD, PhD, ScD5, Pavel G. Shvalb, MD, PhD, ScD5,y,
and Artur A. Isaev, MD1

Abstract
Effective treatment of chronic lower limb ischemia is one of the most challenging issues confronting vascular surgeons. There are a
number of choices available to the vascular surgeon. Open or endovascular revascularization is the treatment of choice when
applicable. Current pharmacological therapies play an auxiliary role and cannot prevent disease progression. Therefore, new
methods of treatment are needed. We conducted a phase 2b/3 multicenter randomized controlled clinical trial of the intramuscular transfer of a plasmid DNA encoding vascular endothelial growth factor (VEGF) 165 with cytomegalovirus promotor
(CMV) in patients with atherosclerotic lower limb ischemia. A total of 100 patients were enrolled in the study, that is, 75 patients
were randomized into the test group and received 2 intramuscular injections of 1.2 mg of pCMV-vegf165, 14 days apart together
with standard pharmacological treatment. In all, 25 patients were randomized into the control group and received standard
treatment only. The following end points were evaluated within the first 6 months of the study and during a 1.5-year additional
follow-up period: pain-free walking distance (PWD), ankle–brachial index (ABI), and blood flow velocity (BFV). The pCMVvegf165 therapy appeared to be significantly more effective than standard treatment. The PWD increased in the test group by

110.4%, 167.2%, and 190.8% at 6 months, 1 year, and 2 years after treatment, respectively. The pCMV-vegf165 intramuscular
transfer caused a statistically significant increase in ABI and BFV. There were no positive results in the control group. Thus, pCMVvegf165 intramuscular gene transfer is an effective method of treatment of moderate to severe claudication due to chronic lower
limb ischemia.
Keywords
chronic lower limb ischemia, VEGF165, gene therapy, clinical trial

Introduction
According to the World Health Organization, cardiovascular diseases are the number 1 cause of death globally.1 Cardiovascular
disease includes both coronary heart disease, cerebrovascular
disease, and peripheral arterial disease, which causes chronic
lower limb ischemia.2 Endovascular or open revascularization
procedures are the main treatment methods for such patients,
although many of them are not suitable for a revascularization
due to severe distal or multifocal atherosclerotic lesions, failed
grafts, or severe coexisting pathology. Thus, new methods to
treat chronic lower limb ischemia should be used.
Along with open surgical, endovascular, and pharmacological
treatment, gene therapy has been introduced to treat patients with
chronic lower limb ischemia. Gene therapy is one of the most rapidly developing methods for treating ischemia.3,4 The following
different types of therapeutic genes that encode various growth
factors have been used in clinical trials: vascular endothelial

1

OJSC ‘‘Human Stem Cells Institute’’, Moscow, Russia
Department of Morphology and General Pathology, Kazan (Volga region)
Federal University, Kazan, Russia
3
Department of Maxillofacial Surgery, A.I. Evdokimov Moscow State University
of Medicine and Dentistry, Moscow, Russia

4
Department of Maxillofacial Surgery, A.I. Burnazyan Medical Biophysical Center,
Moscow, Russia
5
Department of Angiology and Vascular Surgery, Ryazan State I.P. Pavlov
Medical University, Ryazan, Russia
6
Department of Vascular Surgery, Russian National Research Center of
Surgery, Moscow, Russia
7
Department of Surgery, Yaroslavl State Medical Academy, Yaroslavl, Russia
y Deceased
2

Manuscript submitted: October 16, 2014; accepted: January 25, 2015.
Corresponding Author:
Ilia Y. Bozo, 3/2 Gubkina Str, Moscow 199333, Russia.
Email:

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Journal of Cardiovascular Pharmacology and Therapeutics

growth factor (VEGF) 165,5-7 basic fibroblast growth factor,8,9
hypoxia-inducible factor, hepatocyte growth factor,10,11 and others. Gene therapy is designed to induce angiogenesis via the
expression of the aforementioned genes in skeletal muscles after
intramuscular or intravascular delivery of gene products.

In 2010, we completed a phase 1 to 2a clinical trial of
pCMV-vegf165 in patients with chronic lower limb ischemia
(stage 2a to 3 according to Fontaine classification modified by
A. V. Pokrovsky) who were not suitable for reconstructive surgery
or endovascular treatment. This study demonstrated the safety,
feasibility, and short-term (3 months) efficacy of pCMV-vegf165
gene transfer,12,13 which lead to conducting a phase 2b to 3 multicenter clinical trial. The study was conducted under the control of
the Russian Ministry of Health and was completed in 2011.
Patients enrolled in the study were subjected to a 6-month
follow-up period according to the study protocol and an additional
18-month follow-up period for a longer evaluation of study drug
efficacy and safety. The results of the study are reported herein.

Materials and Methods

administered intramuscularly (calf muscles) at 4 to 5 injection
sites in the lower and middle third of the posterior part of the
calf.

Patient Characteristics
The study included patients with chronic lower limb ischemia
who were not suitable for an open or endovascular revascularization due to a severe distal or multifocal atherosclerotic
lesion. The decision was made by a team of vascular surgeons
and radiologists based on the angiographic and echographic
findings, history of the disease, previous procedures, and concomitant pathology. Angiographic score was !7 points according to the Rutherford (1997) runoff classification.
The types of atherosclerotic lesions were defined as follows:
1.

2.

Rationale for the Clinical Study
Preclinical studies of general toxicity (acute, subacute, chronic,
and local irritation) and specific toxicity (allergenicity, reproductive and immune toxicity, mutagenicity, and carcinogenicity) as well as the detection of specific drug activity were
carried out at Russian State Federal Institution ‘‘Institute of
Toxicology of Federal Medical Biological Agency of Russia,’’
Saint-Petersburg (2008). The safety, feasibility, and short-term
efficacy of the study drug were then evaluated in a phase 1 to 2a
multicenter randomized trial that was conducted in 2010 and
enrolled 45 patients.
Federal Service on Surveillance of the Ministry Healthcare
and Social Development of the Russian Federation has granted
the approval to conduct a phase 2b to 3 study (approval notice
No. 177, April 21, 2010). The study protocol was approved by
the National Ethics Committee (protocol No. 62 from April 07,
2010); local ethics committees have also granted their approval
to conduct the study.
All phases of clinical trials were conducted according to the
Declaration of Helsinki of the World Medical Association
‘‘Recommendations guiding physicians in biomedical research
involving human subjects’’ (1964, 2000), ‘‘Rules of Good Clinical Practice in the Russian Federation’’ OST 42-511-99, ICH
GCP rules, and valid regulatory requirements.

Drug Characteristics and Administration Method
The study drug is an original gene construction which contains
a supercoiled plasmid DNA (1.2 mg) encoding pCMV-vegf165
as the active substance and is now marketed as ‘‘Neovasculgen.’’12 The drug was supplied to the study centers as a sterile
lyophilisate that was then dissolved in 2 mL of water for injections immediately prior to administration. The drug was

3.

proximal lesion—patency of proximal arterial segments
(aortoiliac) with a diffuse atherosclerotic lesion (occlusion) of superficial femoral artery and a popliteal artery
extending into the tibioperoneal trunk;
multifocal lesion—patency of proximal arterial segments
(aortoiliac) with a diffuse atherosclerotic lesion (occlusion) of the femoral, popliteal, and both tibial arteries;
distal lesion—patency of proximal arterial segments (aortoiliac, femoral) with a diffuse atherosclerotic lesion
(occlusion) of the popliteal artery with hemodynamically
significant stenosis or occlusion of the tibial arteries.

All patients had a previous history of a long-term moderate
to severe claudication. All patients received aspirin and statins
on a daily basis to reduce the risk of adverse cardiovascular
ischemic events and previously underwent treatment with
pentoxifylline.
Inclusion criteria.
 age more than 40 years;
 a history of stable claudication for at least 3 months;
 stage 2 to 3 chronic ischemia according to Fontaine classification (modified by A. V. Pokrovsky);
 absence of hemodynamically significant stenosis (>70%) of
the aortoiliofemoral arterial segment or (if present) a patent
proximal bypass graft (prosthesis) if revascularization surgery was performed no earlier than 3 months prior to the
inclusion in the study; satisfactory patency of the deep
femoral artery in the presence of hemodynamically significant femoropopliteal arterial lesions;
 presence of hemodynamically significant (stenosis >70%
and/or occlusion) diffuse lesions of the anterior and (or)
posterior tibial arteries (distal lesion);
 voluntary informed consent signed and dated by the patient.
Exclusion criteria.
 chronic lower limb ischemia of nonatherosclerotic genesis (autoimmune disorders, Buerger disease, congenital
abnormalities, vascular injuries, etc);

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Deev et al

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pCMV-vegf165
n=75
Ryazan State
I.P. Pavlov
Medical
University
35/15
Yaroslavl
Regional
Clinical
Hospital
25/5
B.V. Petrovsky
Russian
Scientiϐic
Center of
Surgery 15/5

Control
n=25

Visit 0/1

Visit 2

Visit 3

Visit 4

14 (±2) days 90 (±2) days 180 (±2) days

Screening
Blood and urine laboratory
tests; chest X-rays; abdominal
echography; measurement of
PWD, ABI, BFV;
angiography; SF-36
questionnaire

Treatment Monitoring

1 year

1.5 year

2 years

Follow-up study

Blood and urine laboratory tests, measurement of
PWD, ABI, BFV at each visit; chest X-rays,
abdominal echography, angiography, SF-36

questionnaire – at visit 4

Blood and urine laboratory tests; chest Xrays; abdominal echography;
measurement of PWD, ABI, BFV at each
time point

Figure 1. Design of clinical trial.







stage 4 chronic ischemia according to Fontaine classification
modified by A. V. Pokrovsky (ischemic ulcers and necrotic
lesions);
severe concomitant pathology with life expectancy <1 year;
infectious diseases, history of cancer, or suspected malignancy;
decompensated diabetes mellitus (hemoglobin A1c > 8%
and fasting plasma glucose > 11.1 mmol/L).

Study Design and End Points
The current pCMV-vegf165 gene transfer trial was as an openlabel, prospective, randomized, controlled, and multicenter
study. The patient distribution per study center and time period
is presented in Figure 1.
A total of 100 patients were enrolled in the study and randomized into 2 groups at a ratio of 3:1. Thus, 75 patients were
included into the test group and received 2 injections of
pCMV-vegf165 at a dose of 1.2 mg, 14 days apart (total
dose—2.4 mg) into the calf muscles altogether with standard

pharmacological treatment, and 25 patients were included in
the control group and were given standard therapy alone. All
patients signed and dated the informed consent documents.
In all, 13 patients had ischemic rest pain at baseline: 8 and 5
patients in test and control groups, respectively. In all, 11
patients underwent aortoiliac arterial reconstructive surgery
within more than 6 months prior to the onset of the study (5
patients in the test group and 6 patients in the control group).
In all, 18 patients had compensated diabetes mellitus: 12 in the
test group and 6 in the control group.
In all, 5 patients in the test group and 1 patient in the control
group had undergone limb amputations prior to study enrollment
which did not allow to perform a treadmill test to evaluate painfree walking distance (PWD). The analyzed population in the
study included 94 patients: 70—in the test group and 24—in
the control group. The AP value was variable depending on the
technical equipment of each study site and the period of time
following the study completion, which was considered when
processing statistical data.

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Journal of Cardiovascular Pharmacology and Therapeutics

The initial time points were defined as follows: baseline, 14,
90, and 180 days. Follow-up period was extended to a total of
2 years, with additional time points at 1 and 2 years following

the patients’ inclusion in the study.

Safety Criteria
Safety of pCMV-vegf165 gene transfer in terms of the trial protocol was initially evaluated within 6 months following the
onset of the study (a 14-day in-patient hospital stay, with following out-patient office visits) with the registration of adverse
event (AE) and serious adverse event (SAE) during both routine visits and unscheduled requests for medical care. Moreover, patients who gave their written consent for the
extended follow-up procedures underwent blood and urine
laboratory tests, chest X-rays, and abdominal echography in
order to assess the oncological safety (Figure 1).

Efficacy End Points
Primary Efficacy End Point
Pain-free walking distance. The value of the PWD was defined as
the primary (main) efficacy end point. According to the American College of Cardiology/American Heart Association
Guidelines for the management of patients with peripheral
arterial disease, this value is of the highest importance (class
I recommendations).14 The intragroup distribution of patients
was based on the PWD value. The severity of the disease was
determined according to the Fontaine classification modified
by A. V. Pokrovsky, which is widely accepted in Russia: stage
2a—PWD more than 200 m; stage 2b—less than 200 m, but
more than 50 m; and stage 3—less than 50 m or ischemic rest
pain in absence of ischemic ulcers or necrotic lesions. The
PWD was determined using a treadmill test with reduced initial
speed (1 km/h), as the majority of elderly patients were unable
to perform Gardner test or its equivalents. Information on
patients with ischemic rest pain is also provided (Table 1).

Secondary End Points
Ankle–brachial index. Ankle–brachial index (ABI) was measured

using a standard technique at each visit. Although ABI measurement is regarded as a first-line assessment tool,14 it largely
characterizes main arterial blood flow (macrohemodynamics),
and its diagnostic value is limited in patients who are not suitable for arterial reconstructive surgery due to poor runoff.

Table 1. Baseline Characteristics of Patients.

Factor

Control
Group
(n ¼ 25)

Men, n (%)
Women, n (%)

20 (80.0)
5 (20.0)

pCMV-vegf165 Intergroup
Group
Differences, P
60 (80.0)
15 (20.0)

(Chi-square) 1.000
(Yates corrected
Chi-square)
1.000
(t test) P ¼ .468

Age, mean +
70.9 + 7.8
67.8 + 9.0
SD, years
Severity of chronic lower limb ischemia (stage of disease and rest
pain), n (%)
2a
–
9 (12.0)
2b
22 (88.0)
57 (76.0)
3
3 (12.0)
9 (12.0)
Rest pain
5 (20.0)
8 (10.7)
Occlusion level, n (%)
Proximal
12 (48.0)
38 (50.7)
Distal
5 (20.0)
16 (21.3)
Multifocal
8 (32.0)
21 (28.0)
occlusion
PWD, m

114.3 + 11.4 135.3 + 12.2 (Mann–Whitney
U test with
Bonferroni
correction)
1.000
ABI
0.46 + 0.06
0.51 + 0.02 (Mann–Whitney
U test with
Bonferroni
correction)
1.000
BFV, cm/s
17.6 + 2.1
14.2 + 1.6
(Mann–Whitney
U test with
Bonferroni
correction)
1.000

Abbreviations: ABI, ankle–brachial index; BFV, blood flow velocity; PWD, painfree walking distance; SD, standard deviation.

at 6 months, and 1 year after the onset of treatment. Primary
and repeated angiography was performed using the same
angiography system, by the same radiologist, and with the same
time delay of images. Angiograms were assessed visually by
the same experienced specialist.

Blood flow velocity. Doppler ultrasound techniques are useful in

assessment of lower extremity atherosclerotic lesions and
determining severity of the disease or progression of atherosclerosis.14 Blood flow velocity (BFV) in the posterior tibial
artery was evaluated (if patent).

Quality of life. All patients completed the SF-36 questionnaire
(‘‘SF-36 Health Status Survey’’) before enrollment and at
6 months after the onset of treatment (Figure 1). The following
7 scales were evaluated: physical functioning, physical role functioning, bodily pain, general health perceptions, vitality, social
role functioning, emotional role functioning, and mental
health. The values of each scale varied between 0 and 100, with
100 defined as complete health. All the scales were used to
assess 2 parameters: psychological and physical well-being.

Angiography. Thirty percent of patients enrolled into the study
agreed to undergo a digital subtraction angiography using contrast enhancement at following time points: prior to the study,

Statistical analysis. A sample size of 28 patients in each group
was estimated to detect a 0.75 standardized difference (80%
power, P ¼ .05), assuming the target difference and SD for

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Deev et al

5

PWD to be 75 and 100 m, respectively. We decided to use a 3:1
test/control group ratio in order to make the test group sample
more representative.

The absolute values of efficacy criteria (PWD, ABI, and BFV)
were not normally distributed; therefore, nonparametric methods
were used to test the hypothesis (Mann–Whitney U test and Wilcoxon test with Bonferroni correction to avoid a type I error). The
SF-36 questionnaire scores were normally distributed, so the T
test was used to compare the values of 2 groups.

Results
Baseline Characteristics of the Trial Participants
A total of 100 patients were enrolled in the clinical study: 75
were randomized into the test group and received 2 injections
of pCMV-vegf165, 14 days apart (a total dose of 2.4 mg) into
the calf muscles of the affected limb. The comparison of baseline characteristics between the groups showed that gender differences as well as differences in the primary and secondary
end points were not statistically significant (Table 1). The values of PWD were similar between the 2 groups: 135.3 + 12.2
and 114.3 + 11.4 m in the test and control group, respectively.
A more detailed analysis revealed that the severity of the disease and atherosclerotic lesion levels were comparable among
the control and test groups. However, the control group did not
include patients with stage 2a. Therefore, a precise comparison
between the subgroups regarding the severity of ischemia was
made only in patients with stages 2b to 3 disease.

Evaluation of Safety
No AE, SAE, or significant laboratory abnormalities were
observed in either study group during both treatment and
follow-up period. No peripheral edema was observed. Local
pathological reactions, including allergic, anaphylactic, and
neoplastic reactions, were absent immediately after study drug
administration, at 6 months after the onset of treatment, and
during the extended follow-up period.
During the first 6 months, 3 events precluded the continuation
of the study: 2 acute ischemic strokes (test group) with a positive

outcome and 1 acute myocardial infarction with a fatal outcome
(test group). Apparently, these events were not related to pCMVvegf165 gene transfer as the construction used in the study has a
proven local action. The results of toxicological studies showed
no relationship between the study drug and AEs.15
Tumor growth, eyesight disorders, and other pathological
conditions that could indirectly suggest complications of gene
therapy were not observed in patients throughout the study and
during the 1.5-year follow-up period.

Evaluation of Efficacy
Primary End Point
Pain-free walking distance. The first changes in clinical characteristics among the patients of pCMV-vegf165 group were noticed

by the patients themselves within 2 weeks after the onset of
treatment. More notable changes were observed at 45 to 60
days. The initial PWD level in the test group was 135.3 +
12.2 m, increasing to 284.7 + 29.8 m at 6 months (Tables 2
and 3). The differences between the baseline and subsequent
PWD values within the test group and differences between the
test and control group were statistically significant starting
from day 90. During the first 6 months of the study, there was
an increasing trend of PWD values in 62 (85%) patients of the
test group. During the long-term follow-up period, the value of
PWD continued to increase in the test group. The increase in
the mean distance that a patient could walk without pain was
149.4 m in the study group after 6 months (110.4%), while its
value decreased by 1.5 m in the control group compared to the
baseline. The tendency remained positive throughout the 2
years of monitoring: PWD increased in pCMV-vegf165
patients by 167.2% and 190.8%, that is, by 226.3 and 258.1

m, at 1 and 2 years, respectively, while no statistically significant changes were observed in the control group.
The largest increase in the PWD was observed in patients
with advanced stages of ischemia (severe claudication or
ischemic rest pain), that is, stage 3: a 96.4-m increase
(231.2%) at 6 months, a 228.3-m increase (547.5%) at 1 year,
and a 345.3-m increase (828%) at 2 years. The PWD increased
by 129.4 m (108.3%) in patients with stage 2b disease (initial
PWD increased by 50-200 m). Such positive results remained
stable throughout a 2-year follow-up period. The PWD
increased by 290.0 m (90.6%) in patients with stage 2a disease
at 6 months and by 660.0 m (206.2%) and 517.5 m (161.7%) at
1 and 2 years, respectively.
Depending on the localization of atherosclerotic lesions, the
results were as follows: the 6 months results showed that
patients with multifocal arterial lesions of the lower limbs
benefited from gene therapy. The average increase in PWD values in these patients was 259.0 m (180.7%); the PWD increased
by 431.7 m (301.2%) at 1 year and by 363.7 m (253.8%) at 2
years compared to baseline. The opposite results were obtained
in the control group: PWD increased by 34.0 m (35.4%) at 6
months; however, during the follow-up period, the PWD value
decreased by 56.0 m (À58.3%) and 66.0 m (À68.7%) at 1 and 2
years, respectively, compared to the baseline.
The PWD in test group patients with predominantly distal
vascular lesions increased by 179.7 m at 6 months (132.4%), by
230.3 m (169.7%) at 1 year, and by 342.9 m (252.7%) at 2 years.

Secondary End Points
Characteristics of macrohemodynamics: ABI. Six months following
the onset of the study, there were statistically significant
changes in ABI in the test group (a 0.05 increase, P ¼ .009).

The ABI did not change in the control group. The differences
in absolute values among the test and control groups (patients
with rest pain included) at each visit, as well as an increase in
the absolute values between the groups, were statistically insignificant (Tables 2-4). The long-term follow-up (2 years) results

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135.7 + 22.3

143.3 + 38.3

0.51 + 0.02

14.2 + 1.6

Distal occlusion

Multifocal occlusion

ABI (73/25)

BFV (65/20)

361.6 + 65.8
P ¼ .010

980 + 285.3
P ¼ .243
249.3 + 22.1
P ¼ .240
270 + 36.2
P ¼ .048
216.8 + 38.6
P ¼ 1.000
366.0 + 77.1
P ¼ .135
575.0 + 192.7
P ¼ .056
0.56 + 0.03
P ¼ .009
19.7 + 2.5
P ¼ .010

1.0 Year
393.4 + 66.9
P ¼ .010
837.5 + 392.3
P ¼ .204
286.3 + 39.7
P ¼ .030
387.0 + 88.3
P ¼ .030
260.1 + 38.5
P ¼ .576
478.6 + 105.7
P ¼ .069

507.0 + 176.0
P ¼ .189
0.55 + 0.03
P ¼ .126
24.2 + 2.9
P ¼ .030

2.0 Years

Abbreviations: ABI, ankle–brachial index; BFV, blood flow velocity; PWD, pain-free walking distance.
a
Mean and standard error of the mean.
b
Selection is too small to calculate statistical significance.

122.7 + 11.1

41.7 + 2.9

3

Proximal occlusion

119.5 + 7.9

2b

284.7 + 29.8
P ¼ .010
610 + 129.4

P ¼ .024
248.9 + 23.6
P ¼ .010
138.1 + 19.7
P ¼ .108
210.5 + 24
P ¼ .006
315.4 + 54.7
P ¼ .009
402.3 + 89.3
P ¼ .007
0.56 + 0.02
P ¼ .009
22.6 + 2
P ¼ .010

135.3 + 12.2

320 + 45.9

0.5 Year

Baseline

2a

PWD (70/24)

Value

pCMV-vegf165 Group and Statistical Significance
of Intragroup Differences, M + m

Table 2. Results of Measurements of Primary and Secondary End Points.a

17.6 + 2.1

0.46 + 0.06

96.0 + 16.3

95.0 + 55.0b

133.3 + 32.8

40.7 + 2.3

114.3 + 11.4

114.3 + 11.4

Baseline

130.0 + 23.9
P ¼ .518
0.46 + 0.06
P ¼ 1.000
18.9 + 2.1
P ¼ 1.000

153.3 + 26.0
P ¼ 1.000
100.0 + 50.0b

112.8 + 12.8
P ¼ 1.000
36.7 + 6.7b

112.8 + 12.8
P ¼ 1.000

0.5 Year

–

40.0 + 20.0
P ¼ 1.000
0.49 + 0.08
P ¼ .981
14.8 + 3.2
P ¼ 1.000

.549

.264

0.51 + 0.05b
16.8 + 1.5
P ¼ 1.000

.187

b

.1

1.000

.010

.010

P
0.5 Year

.485

.239

b

b

.104

.353

b

b

b

b

b

b

.080

.010

P
2.0 Years

.280

–

.040

P
1.0 Year

Statistical Significance
of Intergroup Differences, P

30b

125.0 + 75.0b

150b

150b
75.0 + 25.0b

130.0 + 18.2
P ¼ 1.000
–

121.1 + 18.3
P ¼ 1.000

2.0 Years

109.4 + 21.6
P ¼ 1.000
–

109.4 + 21.6
P ¼ 1.000

1.0 Year

Control Group and Statistical Significance
of Intragroup Differences, M + m


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7

Table 3. Results of Measurements of Primary and Secondary End Points.a
pCMV-vegf165 Group, Median, IQR
Value
PWD (70/24)
2a
2b
3
Proximal occlusion
Distal occlusion
Multifocal occlusion
ABI (73/25)
BFV (65/20)

Baseline

0.5 Year

1.0 Year

100, 130
295.0, 185.0
120.0, 100.0
35.0, 25.0
133.0, 132.5
100.0, 145.0
85.0, 90.0
0.50, 0.22

13.7, 12.8

230, 220
525.0, 435.0
230.0, 180.0
80.0, 90.0
210.0, 175.0
305.0, 200.0
340.0, 360.0
0.52, 0.27
20.5, 18.3

230,
1250.0,
177.0,
231.5,
154.0,
300.0,
325.0,
0.55,
18.0,

Control Group, Median, IQR
2.0 Years

258
850.0
237.0
100.0
228.0

300.0
900.0
0.31
14.0

300, 310
525.0, 925.0
300.0, 188.0
400.0, 300.0
220.0, 160.0
400.0, 400.0
400.0, 250.0
0.56, 0.19
20.0, 13.5

Baseline

0.5 Year

1.0 Year

2.0 Years

105, 102.5

140, 110

125, 110

140, 50

142.5, 87.5
30.0, 20.0
150.0, 90.0
100.0, 100.0
150.0, 70.0
0.50, 0.07
20.0, 15.7

125.0, 115.0
–
150.0, 0
75.0, 50.0
40.0, 40.0
0.45, 0.22
14.5, 9.0

120.0,
40.0,
150.0,
95.0,
100.0,
0.50,
19.0,

105.0
8.0
110.0
110.0
50.0

0.08
13.7

145.0,
–
150.0,
125.0,
30.0,
0.50,
16.0,

55.0
0
150.0
0
0.16
4.0

Abbreviations: ABI, ankle–brachial index; BFV, blood flow velocity; PWD, pain-free walking distance.
a
Median and interquartile range (IQR).

Table 4. Mean Values and Standard Error of the Mean (M + m) of ABI and BFV in Patients With Ischemic Rest Pain in Test (pCMV-vegf165) and
Control Groups.
pCMV-vegf165 Group, n ¼ 8

ABI
BFV

Control Group, n ¼ 5

0

0.5 Year

1 Year

2 Years

0

0.5 Year

1 Year

2 Years

0.37 + 0.04
7.1 + 2.7

0.4 + 0.04
16.5 + 51

0.41 + 0.08
15.5 + 8.3

0.37 + 0.11

0.5 + 0.03
11.5 + 3.9

0.53 + 0.05
10.7 + 4.9

0.75 + 0.3
8.5 + 4.5

0.35

Abbreviations: ABI, ankle–brachial index; BFV, blood flow velocity.

demonstrated a slight but stable improvement in ABI in test
group patients.
Characteristics of macrohemodynamics: BFV. The BFV in the
pCMV-vegf165 group patients increased by 8.4 m/s within
6 months (average growth by 59.1%). Within 1 year, the value
of BFV slightly decreased but remained 5.5 m/s higher than the
baseline. At 2 years, the tendency remained positive. There
were no statistically significant changes in BFV in patients of
the control group (Tables 2 and 3).
Angiography. Angiograms were performed and assessed
visually by the same experienced radiologist. Improvement
in the collateral vascular bed was observed in 75% of
patients who agreed to undergo angiography. Enhanced contrast filling of the microcirculatory bloodstream due to an
increased diameter of collateral vessels was recorded in
12.5% of patients who underwent angiography. A moderate
increase in the number of the newly formed collaterals was
recorded in 37.5% of patients. Significant growth of the collateral vessels was registered in 37.5% of patients who
underwent angiography. Neoangiogenesis may be attributed
to the growth of new collaterals and possibly to the opening

of previously nonfunctioning vessels (Figure 2). There were
no clinically important laboratory abnormalities throughout
the entire period of treatment and follow-up in patients of
both groups.

Figure 2. Angiographic images, patient of the test group: (A) before
treatment and (B) 6 months after pCMV-vegf165 gene transfer.

Quality of life. A statistically insignificant improvement in physical health was observed in patients of the test group. Mental
health also slightly improved in patients of the pCMV-vegf165
group (Table 5). Control patients had a higher quality of life
regarding mental health compared to patients who were treated
with gene transfer.

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Journal of Cardiovascular Pharmacology and Therapeutics

Table 5. Values of Physical and Mental Health in Patients of Test (pCMV-vegf165) and Control Groups According to SF-36 Questionnaire.
Statistical Significance of Intergroup Differences, P (T test)
Value

Group

Physical health

pCMV-vegf165 group

Control group
pCMV-vegf165 group
Control group

Mental health

Baseline, M + m
36.0 +
36.7 +
35.1 +
44.5 +

1.3
2.9
2.6
4.3

0.5 Year, M + m
38.5 +
36.4 +
39.9 +
46.9 +

Discussion
The concept of gene therapy for paracrine vascular growth regulation, that is, therapeutic angiogenesis, began evolving after
the pioneering works of Isner.5,16,17 Gene therapy evolved due
to the accomplishment of experimental and clinical trials which
investigated different therapeutic genes.3 A number of delivery
vectors were used: viral (mainly adenoviruses)3,7 and nonviral
(mainly naked plasmids).3,5,6,18 Majority of clinical trials

demonstrated safety of both approaches of local administration
of gene products at different dosage levels in terms of systemic
allergic or anaphylactic reactions and the absence of neoplastic
reactions, for example, proliferative retinopathy, vascular tumors,
induction of dormant tumors, and so on.3,18 However, data regarding the efficacy of gene therapy were more variable. Certain studies were considered a failure due to the chosen requirements
regarding efficacy end points, such as the number of amputations
or the survival curve,19 heterogeneity of patients enrolled into the
study, and selection of a therapeutic gene, for example, not the
most promising candidate genes for angiogenesis.
Present study aimed to determine the safety and efficacy of
the pCMV-vegf165 gene product in patients who were not suitable for surgical or endovascular revascularization. The absence
of ischemic ulcers and necrotic lesions in patients (stage 4
according to Fontaine classification modified by A. V. Pokrovsky) allowed to study the effect of gene transfer in patients
with viable limbs. Majority of previous studies enrolled patients
with ulcers or gangrenes, which had a negative impact on further
investigations of gene products and their effects or use in
patients with moderate to severe claudication.19-21
Within the study (180 days) and follow-up period (another
1.5 years) neither of 3 study centers reported any adverse effects
(AEs and SAEs) or other complications. The selected mode of
pCMV-vegf165 administration at the selected dosage regimen
was safe during the therapy and at least 2 years thereafter.
All lethal outcomes (5 in the test group and 2 in the control
group) were attributed to acute myocardial infarction (Table 6).
Peripheral arterial disease is an independent predictor of worse
outcomes in patients with ischemic heart disease. We believe that
there is no relationship between lethal outcomes and gene transfer
in terms of this study. These findings correspond with the results
of other studies of plasmid VEGFf165 gene products.3,17,22
The evaluation of efficacy appeared more difficult. The use

of gene therapy in patients who were not suitable for an open or
endovascular revascularization allowed significant increases in
PWD. This positive tendency was stable both during the first

3.1
2.4
2.3
4.1

Baseline

0.5 Year

.037

.241

.028

.001

Table 6. Number of Amputations and Patients Who Died During the
Observation Period in Test (pCMV-vegf165) and Control Groups.
pCMV-vegf165 Group

Time Points

Control Group

Patients

Patients
Who
Who
Died
Died
Amputations
Amputations

6 Months
From 6 months
to 1 year
From 1 to 2 years

0
5a

2
3

0
2b

0
1

0

0

1

0

a

Four amputations were performed in patients with ischemic rest pain at
baseline.
b
One amputation was performed in patients with ischemic rest pain at baseline.

Figure 3. Patient intergroup ratio according to the stage of disease at
baseline, at 0.5, and 1 year after administration: (A) test group (pCMVvegf165) and (B) control group. I—stage 2a; II—2b; III—stage 3; and
black—amputations.

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Deev et al

9

6 months and the following 1.5 years. The PWD continued to
increase at the end of the 2-year monitoring period. Improvement in PWD in the test group was compared to the changes
in PWD in the control group: while the conventional therapy
alone did not have major successful results, gene transfer had
statistically significant positive effects (Tables 2 and 3). Unfortunately, our data cannot be compared to the results of similar
studies, as most of the previous studies enrolled patients with
critical lower limb ischemia who could not undergo the treadmill exercise testing.7,8,11,23,24 Although the study design (not
blinded, not placebo-controlled) contributed to comparison difficulties, we were able to notice that the number of test group
patients with less severe stage of the disease increased, while

a negative tendency was observed in the control group due to
an increased number of patients with more advanced stages
of the disease, including those which resulted in amputation
(Figure 3). At 1 year following the onset of treatment, 4 of the
total 5 amputations in the test group were performed in patients
with pain at rest at baseline (Table 6). Of a total of 2 amputations in the control group, 1 was performed in a patient with
ischemic rest pain at baseline. Limb loss was attributed to the
disease progression leading to irreversible ischemia. Patients
enrolled in the study were not suitable for revascularization,
consequently performing an amputation was the only option
left.
Limb salvage rates at 2 years were 93.3% in the test group
and 88% in the control group (Table 6). However, the differences were not statistically significant. So, we did not observe
the amputation reduction in the test group. More observations
are needed.
Clinical signs majorly improved in patients with distal or
multifocal atherosclerotic lesion.
Changes in ABI and BFV were not significant which may be
explained by the fact that the study drug is designed to induce
angiogenesis at the microcirculatory level and does not affect
macrohemodynamic. Nevertheless, slight improvement in ABI
and BFW may be attributed to the general improvements in the
collateral arterial flow and decrease in the peripheral arterial
resistance.
Safety and efficacy of the studied pCMV-vegf165 gene
product marketed as ‘‘Neovasculgen’’ were demonstrated in
selected patients throughout a 2-year follow-up period.15
Within this period, we were able to track both limb salvage and
patient survival (Table 6). Gene therapy with pCMV-vegf165
did not affect mortality. Limb salvage largely depended on the

presence of rest pain at baseline. However, these parameters
should be analyzed in larger cohorts of patients.
Despite the marked improvement in claudication symptoms
in the test group patients, gene therapy did not significantly
affect the quality of life. Mental health score was higher in the
control group as compared to those of the test group. Apparently, these findings were attributed to the initial differences
in baseline SF-36 scores among the patients of both groups
(Table 5) and presence of concomitant pathology which
decreased the positive impact of PWD increase in the overall
quality of life. Such findings may indicate the presence of an

underlying depressive disorder in patients with chronic lower
limb ischemia.

Conclusion
The use of the plasmid DNA gene product encoding VEGF165
(pCMV-vegf165) in combination with standard pharmacological therapy significantly improves clinical signs of claudication
in patients with chronic lower limb ischemia. A 2-year followup demonstrated a stable PWD improvement. The results of
the study were sufficient for the registration of ‘‘Neovasculgen’’ as a drug which is used in the treatment of patients with
moderate to severe claudication due to stage 2a to 3 atherosclerotic chronic lower limb ischemia. However, further studies
enrolling larger groups of patients are needed to completely
evaluate the effects of pCMV-vegf165 gene transfer in patients
with pain at rest due to peripheral atherosclerosis, ischemia
caused by diabetes mellitus or autoimmune disorders, and those
who undergo peripheral arterial revascularization.
Author Contributions
R. Deev contributed to design, contributed to analysis and interpretation, drafted the article, critically revised the article, and gave final
approval. I. Bozo contributed to analysis and interpretation, drafted
the article, critically revised the article, and gave final approval.
N. Mzhavanadze contributed to acquisition and analysis, drafted the

article, critically revised the article, and gave final approval. D. Voronov
contributed to acquisition and analysis, critically revised the article,
and gave final approval. A. Gavrilenko critically revised the article
and gave final approval. Y. Chervyakov contributed to acquisition,
critically revised the article, gave final approval, and agrees to be
accountable for all aspects of work ensuring integrity and accuracy.
I. Staroverov contributed to acquisition, critically revised the article,
and gave final approval. R. Kalinin contributed to acquisition, critically revised the article, gave final approval, and agrees to be accountable for all aspects of work ensuring integrity and accuracy. P. Shvalb
contributed to conception and design, contributed to acquisition, critically revised the article, and gave final approval. A. Isaev contributed to conception and design, contributed to analysis and interpretation,
critically revised the article, and gave final approval.

Acknowledgments
Authors would like to thank Prof S. L. Kiselev for his contribution in
developing the gene construction and participating in the studies.

Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest
with respect to the research, authorship, and/or publication of this article: A. A. Isaev, I. Ya. Bozo, and R. V. Deev are employees of the
OJSC ‘‘Human Stem Cells Institute.’’ A. A. Isaev is shareholder of the
OJSC ‘‘Human Stem Cells Institute.’’

Funding
The author(s) disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: OJSC
‘‘Human Stem Cells Institute’’ (Moscow, Russia) sponsored the clinical trial.

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Journal of Cardiovascular Pharmacology and Therapeutics

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