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  • Safety of SeV Vector

On the Safety of Sendai virus vector

[1] Sendai virus vector: Distinctive features
Sendai virus vector is a versatile vector developed in Japan from Sendai virus, a murine parainfluenza virus of the paramyxovirus family (1). Sendai virus has a single-stranded RNA molecule as the genome. The genome remains in the cytoplasm of the infected cell throughout the replication cycle of the virus and is never converted to DNA because Sendai virus replication does not require transfer of the genome to the nucleus or integration into the chromosome. Sendai virus vector is based on the Sendai virus backbone and have the following distinctive features.

1. No disturbance of host genetic information
Sendai virus vector, a cytoplasmic RNA virus vector, has a level of safety unparalleled by other vectors that have been used to date (2). This high level of safety stems from the fact that the entire life cycle of Sendai virus vector, including replication of the genome and expression of the encoded genes, is carried out in the RNA form exclusively in the cytoplasm and the virus does not integrate into the host chromosome, thus never disturbing the genetic information on the host chromosome (Fig.1). In this sense, vectors based on retroviruses and lentiviruses, similarly having RNA genome, are totally different from Sendai virus vector because the genome of retroviruses and lentiviruses is converted to DNA in the infected cells and integrated into the host chromosome before the genetic information is transcribed and expressed.
It has been reported that some of the patients that had received a retrovirus vector-based gene therapy for the treatment of X1-linked severe combined immunodeficiency (X-SCID) in France in 2002-2005 developed leukemia due to integration of the vector in the proximate region of an oncogene in the chromosome of these patients (3). In contrast, such a risk, in principle, does not exist with Sendai virus vector. Moreover, even if administered Sendai virus vector spreads systemically and, by any chance, reaches the reproductive cells, there will be no genetic change that can be carried over to the next generations because of the lack of recombination with the chromosome.
In a recent study, it has been revealed that vectors based on DNA, such as adenovirus vector, adeno associated virus (AAV) vector, plasmid vector, integrate into the host chromosome although at a low frequency.
Therefore, the non-disturbing nature of Sendai virus on the host genetic information is considered to be a principal safety advantage over other vectors, including DNA vectors (7).
Fig.1 The life cycle of Sendai virus

2. High expression level
Sendai virus vector has an overwhelmingly high level of gene expression compared with other vectors in use, including plasmid vectors, and thus a small amount of Sendai virus is enough to achieve expression of various proteins to a desired concentration locally for a period of time. For example, Sendai virus vector achieved a higher expression level in cultured cells, respiratory tissues, blood vessel walls, and skeletal muscles than adenovirus and plasmid vectors (4,5,6,7). When administered to mouse skeletal muscles at the relatively low titer of 1x107 infectious particles/mouse, Sendai virus achieved a gene expression level 10 times higher than that could be achieved with a plasmid vector administered at a dose (100 μg/mouse) 50 times higher than the dose clinically used in the US and Europe.
Such powerful capability of gene transfer and expression plays an important role in the safety of Sendai virus-based gene therapeutics and vaccines. In the gene therapy accident of 1999 at the University of Pennsylvania in the US, it was pointed out that the main cause of accident was the severe innate immune reaction and hypercytokinemia (for example, IL6 > 6,000 pg/ml) induced by the unusually large amount of adenovirus vector, 1x 1013 particles, infused to the ornithine transcarbamylase (OTC) deficiency patient, who died after the treatment (8).
In contrast, it was reported in a preclinical study that Sendai virus vector achieved the highest efficiency of gene expression at 1x109-1x1010 active particles per person (calculation based on animal-human weight ratio), which was 1/1,000-1/10,000 of the adenovirus vector used in the above accident. Therefore the possibility of Sendai virus inducing dangerous reactions such as hypercytokinemia is considered remote. In fact, in the phase I/IIa clinical study conducted at Kyushu University Hospital, the maximum blood level of IL6 was only 13.6 pg/ml in patients who received 1x109 active particles of Sendai virus.
In sum, the powerful gene transfer and expression capability of Sendai virus enables the vector to achieve desired effect with a small dosage, and thus brings the high safety of the vector.

3. No reported pathogenicity in humans
To date, there has been no report of Sendai virus having pathogenicity in humans. Since the discovery in the 1950s, Sendai virus has been continuously used in the laboratory but there has been no report of accidental infection of humans by Sendai virus. DNAVEC's Sendai virus vector is based on Sendai virus Z strain, a greatly attenuated laboratory strain with a long history of passage in the laboratory. In addition, in the recent phase 1 clinical trial by St. Jude Children’s Research Hospital in the US using a wild-type Sendai virus, no adverse events due to Sendai virus administration were reported (9, see below).

4. Nontransmissible Sendai virus vector is available
ID Pharma has developed a Sendai virus vector rendered “nontransmissible” by deleting the entire gene of the envelope protein F, which is an essential component necessary for infection, from the genome (10). The F protein is supplied to the vector particle in trans from a helper cell line, constructed to express F protein to a high level, in the manufacturing process of the “nontransmissible” vector, enabling the vector to infect target cells using the F protein supplied to the envelope surface. However, there will be no supply of F protein in the infected cells, and therefore the particles generated in the infected cells are incapable of infecting surrounding cells; thus transmission of the vector from cell to cell is impossible. This genetic modification gives added safety to Sendai virus vector without sacrificing the high expression capability of the vector.
Because no recombination occurs between RNA molecules, there is theoretically no chance of F-deleted “non-transmissible” Sendai virus vector reverting to a transmissible virus by obtaining a replacement F gene by recombination with other parainfluenza viruses.
Fig.2 gives the genome structure of the wild type and F-deleted “nontransmissible” type Sendai virus vectors. To use the vectors for gene therapeutics and vaccines, the gene of interest will be inserted upstream of the NP gene near the 3’ terminal of the RNA genome.

Fig.2 Genome structure of Sendai virus vector

[2] Preclinical study of Sendai virus vector
ID Pharma has conducted a comprehensive preclinical studies of the Sendai virus vector based therapeutic DVC1-0101 for the treatment of peripheral artery disease (PAD). The results of the studies are summarized below:

1. Toxicology
1) Single dose acute toxicity study
DVC1-0101 was injected to the hind limb muscles of mice, the natural host of Sendai virus, and examined pathologically and histologically for acute toxicity. No abnormal conditions were observed in parameters including clinical symptoms, hematology, blood chemistry, or organ weight. Histopathology revealed minor inflammatory reaction at the injection site, but inflammation receded to normal by day 14 after dosing. In studies using rats of both sexes injected DVC1-0101 intramuscularly or intravenously, no significant adverse events were observed at the maximum dose physically possible (60 times the planned maximum clinical dose for human). No changes were observed in the level of various cytokines. In monkeys intramuscularly injected with DVC1-0101, no toxicity was observed except for minor inflammation in the muscles at the injection site.

2) Repeat dose toxicity studies
Two week repeat dose toxicity studies (intramuscular administration of DVC1-0101 daily for 14 days) were conducted using rats and monkeys of both sexes. In the monkey study, reactions such as minor vacuole formation and slight infiltration by inflammatory cells were observed at the site of injection by the examination on the second day after dosing but these reactions disappeared after the observation period and were considered transient, reversible changes. In the rat study, no toxicological reactions were evident except for minor reaction to the vector.

3) Carcinogenicity study
Short-term carcinogenicity studies (2-week repeated intramuscular administration with 32 weeks of observation) using tumor-prone mouse strain indicated no carcinogenic effect with DVC1-0101.

4) Tissue damage studies
DVC1-0101 was administered into the femoral muscle of mice in a single dose. In the initial stages of administration, mild inflammatory cellular infiltration was observed in the interstitial section of the muscle bundle at the site of administration, as well as in the surrounding fat tissue. However, these changes disappeared almost completely after 2 or 3 weeks. No changes such as necrosis of muscles were observed.

2. Single dose biodistribution studies
In vivo kinetics of DVC1-0101 after single intramuscular administration was investigated for male and female rats by the qualitative reverse transcription PCR (qRT-PCR) method. Vector genome was detected in the muscles at the injection site only during the early stage after administration. After intravenous injection to rats, vector genome was detected in the heart, lungs, blood and spleen only during the early period after administration (for 1 week at the maximum). In a study using mice, only the muscles at the injection site were positive for vector genome early after administration (days 2 and 7) but all animals became negative for vector genome from day 14 onward. From these results, it was concluded that the administered DVC1-0101 transiently distributed almost exclusively in the muscles at the site of administration.

[3] Safety evaluation in clinical studies
1. Phase 1 clinical trial of wild-type Sendai virus (St. Jude Children’s Research Hospital)
The purpose of this trial was to test wild-type Sendai virus as a live vaccine against human parainfluenza 1 intended for use in infants (9). In this study, 9 healthy adults (2 males, 7 females, average age 28.6 years old) were intranasally administered with wild-type Sendai virus in 3 increasing doses. In 2 weeks after administration, marked elevation of anti-parainfluenza 1 antibody level was observed in 3 of 9 subjects, indicating effectiveness of the vaccine. None of the subjects showed respiratory symptoms and hematology tests (12 items including numbers of neutrophils and monocytes) indicated no abnormality. From these results it was confirmed that wild-type Sendai virus was well tolerated by the subjects. The authors also reported that, in a preclinical study using monkeys, they observed no respiratory symptoms after intranasal administration (11).

2. Clinical study of DVC1-0101 by Kyushu University
In the clinical study titled “Clinical study for angiogenic gene therapy to treat chronic critical limb ischemia (arteriosclerosis obliterans and Buerger's disease) via non-transmissible recombinant Sendai virus vector carrying angiogenic factor (fibroblast growth factor 2) gene,”12 patients were administered to patients of chronic critical limb ischemia with increasing doses of DVC1-0101 (3 patients/dose x 4 doses). The major endpoint of this study was to determine the safety of DVC1-0101. No severe adverse events causally related to the administration of DVC1-0101were observed. An official safety evaluation committee evaluated the results and concluded that DVC1-0101 was safe for use in critical limb ischemia patients because DVC1-0101 was highly tolerated and its systemic effect was small (12).

[4] Conclusion
Sendai virus vector, a cytoplasmic RNA virus vector, has a high level of safety unmatched by other vectors used to date. In fact, DVC1-0101, the frontrunner of Sendai virus vector based therapeutics and vaccines, no severe adverse effects were observed in both preclinical and clinical studies. In addition, a clinical trial of an AIDS vaccine based on Sendai virus vector is currently conducted by the US NPO International AIDS Vaccine Initiative (IAVI) in Africa and Europe with healthy volunteers, and so far no adverse events have been reported. It can be concluded from the above safety data that have been accumulated over the years that Sendai virus vector is safe for use in humans.

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