4. Clinical Trials
Despite being a relatively young field and the set-backs that were experienced, an impressively large number of clinical trials have been conducted and an increasing number of trials are planned or in progress. A summary of trials is presented below and in
Table 3.
The classic example of retrovirus-based gene therapy trials relates to the treatment of children with SCID, which provided complete cure although development of leukemia occurred in some patients [
3,
4]. Since then, thorough vector development related to safety and efficacy issues has revitalized the application of retroviruses for clinical trials. For instance, Toca 511 retrovirus vectors have been subjected to a clinical phase I trial in patients with recurrent high-grade glioma (HGG) [
122]. The overall survival rate of 13.6 months was statistically better than for the control group. A phase II/III clinical trial on HGG patients is currently in progress [
123]. Gammaretroviral vectors have been evaluated in a phase I/II trial in patients with chronic granulomatous disease (CGD) characterized by primary immunodeficiency, resulting in impaired antimicrobial activity in phagocytic cells [
124]. The results indicated that despite transiently resolved bacterial and fungal infections, clonal dominance and malignant transformations compromised therapeutic efficacy.
Lentivirus-based clinical trials have been less common than those for retroviruses due to their later vector development. For instance, cystic fibrosis has recently been targeted by lentivirus vectors pseudotyped with F/HN showing efficient gene transfer to lungs [
125]. In preparation for clinical trials promoter/enhancer elements were assessed in mice and human air-liquid interface cultures, transduction efficiency determined, and integration site profiles were mapped. The optimized lentivirus carried a hybrid cytosine guanine dinucleotide (CpG)-free enhancer/elongation factor 1 alpha promoter (hCEF) and the cystic fibrosis membrane conductance receptor (CFTR) as the therapeutic gene, providing 90% transduction efficiency in clinically relevant delivery settings and expression of functional CFTR. These findings support the initiation of the first-in-man trial in CF patients. Lentivirus-based gene therapy has been applied for two phase I/II studies in patients with transfusion-dependent β-thalassemia [
126]. Mobilized autologous CD34
+ cells from 22 β-thalassemia patients were transduced ex vivo with the LentiGlobin BB305 vector expressing the adult hemoglobin (HbA) gene with a T87Q amino acid substitution. The lentivirus treatment reduced or eliminated the needs for long-term red cell transfusions in 22 patients with severe β-thalassemia without any serious adverse events related to the drug. Lentiviruses have found applications in treatment of leukemia. In this context, a VSV-G pseudotyped lentivirus (pELPs 19-BB-z) vector expressing a chimeric antigen receptor with specificity for the B cell antigen CD19. A low dose of autologous chimeric antigen receptor-modified T cells (1.5 × 10
5 cells/kg body weight) reinfused into a patient with refractory chronic lymphocytic leukemia (CLL) persisted at high levels for 6 months in the blood and bone marrow [
127]. Furthermore, remission continued for 10 months. Moreover, the lentivirus-based chimeric antigen receptor-modified T (CAR-T) cells targeting CD19 therapy was evaluated in 30 children and adults with relapsed acute lymphoblastic leukemia (ALL) [
128]. Moreover, the lentivirus-based CAR-T therapy was recently approved by the US FDA as CTL019 or tisagenlecleucel for refractory/relapsed ALL [
129]. Stem cell-based lentivirus gene therapy applied for hemophilia A treatment demonstrated life-long production of factor VIII (FVIII) and cure of disease [
130]. Moreover, lentivirus-based transduction of stem cells differentiated to adipogenic, chondrogenic, and osteoblastic cells provided high level expression of factor IX applicable for hemophilia B treatment [
131]. In attempts to target PD, the lentiviral-based gene therapy approach ProSavin®, the approach was to restore local and continuous dopamine production by delivery of three enzymes in the dopamine biosynthesis pathway [
132]. In a Phase I/II clinical trial, patients with advanced PD showed some improvements in motor behavior. However, higher levels of dopamine replacement required for increased benefits of the treatment might be achieved by optimizing the gene order in the ProSavin® expression cassette and by engineering fusions of two or three of the transgenes. These approaches resulted in enhanced dopamine and L-Dopa production. Furthermore, the equine infectious anemia virus (EIAV)-TCiA showed significantly improved dopamine and L-Dopa production compared to ProSavin® in human neuronal cells, demonstrating expression of all three enzymes. Next, clinical evaluation of EIAV-TCiA will be conducted in PD patients. Lentivirus vectors have also been applied for development of HIV therapy by shRNA delivery targeting CCR5 [
133]. Delivery of shRNAs effectively inhibited CCR5 expression resulting in protection against HIV-1 infections in cell cultures [
134]. Engineering of a self-inactivating lentivirus vector expressing the sh5 anti-HIV gene and the C46 anti-viral fusion inhibitor peptide contributed to a synergistic effect on HIV-1 inhibition [
133]. Moreover, the first clinical trial for lentivirus-based RNAi (LVsh5/C46) conducted in HIV patients demonstrated safety and protected the immune system from the effects of HIV without using anti-retroviral drugs [
135].
In the context of alphaviruses, VEE particles expressing the CMV gB or PP65/IE1 fusion protein were evaluated in a randomized, double-blind Phase I clinical trial in CMV seronegative individuals [
136]. The well-tolerated intratumoral or subcutaneous administration showed no clinically important changes, direct IFN-γ enzyme-linked immune absorbent spot (ELISPOT) responses to CMV antigens were detected in all 40 vaccinated subjects, and neutralizing antibody and multifunctional T cell responses against all three CMV antigens were obtained. Moreover, healthy HIV-negative volunteers were subjected to subcutaneous immunization with VEE particles expressing a nonmyristoylated form of HIV Gag in Phase I trials in the United States and South Africa [
137]. Although the treatment was well tolerated, only modest local immune responses with low levels of binding antibodies and T cell responses was achieved. Dendritic cell (DC)-targeting VEE particles expressing CEA were subjected to intratumoral doses of 4 × 10
7 to 4 × 10
8 IU recombinant particles administered every 3 weeks four times in patients with advanced cancer [
138]. Repeated immunization elicited clinically relevant CEA-specific T cell and antibody responses and prolonged survival was obtained in patients with CEA-specific T cell responses. A Phase I trial on VEE particles expressing the prostate-specific membrane antigen (PSMA) was carried out in patients with castration resistant metastatic prostate cancer (CRPC) [
139]. CRPC patients administered with either 0.9 × 10
7 IU or 3.6 × 10
7 IU showed good tolerance of both doses although the detected PSMA-specific signals were weak. Despite that neither robust immune responses nor clinical benefits were obtained, the presence of neutralizing antibodies indicated that dose optimization might improve the immunogenicity. In attempts to provide passive tumor targeting, SFV particles were encapsulated in liposomes [
24]. Intravenous administration of liposome encapsulated SFV particles expressing IL-12 (LipoVIL12) in kidney carcinoma and melanoma patients showed transient 10-fold enhanced IL-12 plasma levels in a phase I trial. Moreover, the encapsulation substantially enhanced tumor targeting and prevented recognition by the host immune system after repeated administration.
Clinical trials for rhabdoviruses have mainly been comprised of immunization studies for VSV vectors targeting infectious diseases, particularly EBOV. In this context, VSV vectors expressing the glycoprotein of the EBOV Zaire strain (ZEBOV) were subjected to a placebo-controlled, double-blind, dose-escalation Phase I trial in 78 volunteers receiving three doses of 3 × 10
6, 2 × 10
7, or 1 × 10
8 pfu of VSV-ZEBOV [
140]. Although some adverse events such as injection site pain, fatigue, myalgia, and headache occurred the overall safety profile was good. The lowest dose elicited lower antibody titers at day 28 compared to the two higher doses. Significantly increased antibody titers were observed after a second immunization on day 28, but the effect disappeared after six months. In another placebo-controlled, randomized, dose-ranging, observer-blind Phase I trial the attenuated VSVΔG- doses with sustainable IgG titers were obtained in all 40 participants [
141]. Moreover, the previously applied doses of 1–5 × 10
7 pfu were reduced to 3 × 10
5 pfu in the dose-finding, placebo-controlled, double-blind Geneva Phase I/II study, which improved tolerability, but decreased antibody responses [
142]. The superiority of the VSV-based (VSVΔG-ZEBOV-GP) vaccine in comparison to the chimpanzee adenovirus 3 (ChAd3-EBO-Z) was demonstrated in a randomized, placebo-controlled Phase III trial in 1500 adults in Liberia [
143]. Adverse events such as injection site reactions, headache, fever, and fatigue were more common in individuals receiving active vaccine in comparison to placebo. Superior antibody responses one month after immunizations were obtained for VSVΔG-ZEBOV-GP (83.7%) compared to ChAd3-EBO-Z (70.8%), and placebo (2.8%). After 12 months, VSVΔG-ZEBOV-GP immunization generated better antibody responses (79.5%) than vaccination with ChAd3-EBO-Z (63.5%) and placebo (6.8%). A Phase III open-label, cluster-randomized ring vaccination trial was conducted in 7651 suspected EBOV cases in Guinea [
144]. Among the participants 4123 individuals were assigned for immediate vaccination with VSV-EBOV and 3528 persons for delayed vaccination. No cases of EBOV were detected in the group receiving the vaccine at the start of the study after ten days, whereas 16 EBOV cases were discovered in the individuals receiving delayed vaccination. Overall, a good safety profile was associated with the VSV-ZEBOV vaccine also providing promising protection against EBOV. Moreover, a Phase III randomized clinical trial was conducted in 4160 individuals in Guinea and Sierra Leone [
145]. A dose of 2 × 10
7 pfu of VSV-ZEBOV was administered at the start of the trial to 2119 individuals and 2014 persons were immunized after a delay of 21 days. Substantial EBOV protection was achieved during the follow-up period of 84 days and no new cases of EBOV were detected after day 10. In another individually controlled Phase II/III clinical trial, health care and frontline workers in the five most EBOV affected districts in Sierra Leone were subjected to a single intramuscular injection of VSV-ZEBOV which was administered at enrollment or 18–24 weeks later [
146]. Because of the low case frequency as the EBOV epidemic was controlled, neither EBOV cases nor vaccine-related serious adverse events were reported.
Measles viruses have been subjected to clinical trials mainly in cancer therapy. For instance, an open-label, non-randomized, dose-escalation Phase I trial was conducted in patients with cutaneous T cell lymphomas with the unmodified MV-Edm Zagreb (MV-EZ) strain [
147]. Intratumoral injections of MV-EZ on days 4 and 17 preceded by subcutaneous IFNα injections (24 and 72 h earlier) defined the maximum tolerated dose as 10
3 TCID
50. The MV-EZ treatment resulted in complete regression of cutaneous T cell lymphoma (CTCL) lesions in one patient and partial regression in the other patients. Moreover, a Phase I trial in patients with advanced ovarian cancer was performed by intraperitoneal injection of 10
3–10
9 MV-CEA showing no dose-limiting toxicity [
148]. Stable disease was observed in 14 patients with a median duration of 88 days and a range of 55–277 days. Related to the applied dose, 10
7–10
9 TCID
50 resulted in stable disease in all patients, whereas it was accomplished for only five out of 12 with doses between 10
3 and 10
6 TCID
50. MV-CEA has also been planned for a Phase I trial in patients with recurrent glioblastoma multiforme, where escalating doses from 1 × 10
5 to 2 × 10
7 TCID
50 will be administered either into the resection cavity or into recurrent tumors [
149]. Until now, three patients receiving 1 × 10
5 TCID
50 and three other patients receiving 1 × 10
6 TCID
50 showed no dose-limiting toxicity. Patients with relapsed refractory myeloma have been subjected to a Phase I clinical trial of intravenous administration of oncolytic MV vectors expressing the human sodium iodide symporter (NIS) [
150]. As the original dose-escalation study did not reach the maximum tolerated dose (MTD), doses of 1 × 10
10 and 1 × 10
11 TCID
50 were tested resulting in a complete response persisting for nine months in one patient with the higher dose, where after an isolated relapse occurred in the skull without recurrent marrow involvement. The patient remained disease-free for an additional 19 months due to an irradiation procedure.
Newcastle disease virus have been subjected to several clinical trials in the area of cancer. For instance, expression of multiple tumor-associated antigens (TAAs) from NDV vectors has provided long-term survival in Phase II trials in ovarian, stomach, and pancreatic cancers [
151]. In another study, the NDV PV101 strain was intravenously administered to 79 patients with advanced solid tumors in a Phase II trial at a low dose of 1.2 × 10
10 pfu/m
2 and a high dose of 1.2 × 10
11 pfu/m
2 [
152]. Administration of the higher dose resulted in objective responses and progression-free survival ranging from four to 31 months. In contrast, a randomized double-blind Phase II/III trial in melanoma patients provided no remarkable differences between individuals vaccinated with NDV and the placebo group [
153]. In a more positive outcome, 335 patients with colorectal cancer were immunized with NDV vectors in a Phase III trial, which prolonged survival and improved short-term quality of life in patients [
154].
Coxsackievirus vectors have been applied for clinical trials mainly for the treatment of melanoma. In a Phase I/II trial melanoma patients treated with CVA21 demonstrated good tolerance, viral replication in tumors, and increased antitumor activity [
155]. Moreover, coxsackievirus vectors have been subjected to combination therapy. In this context, the antitumor activity of CVA21 was enhanced by co-treatment of melanoma patients with immune checkpoint blockade in a Phase II trial, which led to induced immune cell filtration in the tumor environment [
156]. In a Phase 1b clinical trial in melanoma patients, combination therapy of CVA21 and systemic administration of pembrolizumab showed a best overall response rate of 60% and stable disease in 27% of the patients [
157].