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ACHIEVEMENTS IN VECTOR DESIGN

The successful realization of gene therapy programs in medicine is highly dependent upon the degree of vector design development. This area of investigation has to deal with a number of pressing and complex issues in order to optimize the performance of gene transfer technology in preclinical studies and clinical research. The aspects that need to be addressed may be summarized as follows:

• The transduction efficiency of both viral- and nonviral-based vectors must be improved. Also, the production and purification procedures for vectors must be optimized.
• In the matter of gene delivery safety, the first rule is that vectors must not be pathogenic or toxic to the patients. For this reason, viral vectors have been engineered to be noncompetent for replication, and devoid of viral factors that may pose a hazard in humans. However, a great deal of attention is still drawn to the possibility of replication-competent virus formation in patients. Another concern is the issue of insertional mutagenesis of vectors based on retroviruses or on adeno-associated virus (AAV) type 1 or type 2. A rather new aspect that has been considered is the possible recombination between retroviral-based vectors and human endogenous retroviruses (HERVs). In order to improve the performance of gene transfer technology, viral-based vectors must be modified in order to reduce their toxicity and immunogenicity in patients. A number of significant advances have been accomplished in this respect. One study has also raised some concern about the immunogenicity of selectable markers [54], which normally derive from bacteria. Therefore, the transduction of cells of the hematopoietic lineages may lead to selectable markers entering the antigen-presenting cell pathway. This in turn would render the transduced cells susceptible to cytotoxic T lymphocyte (CTL) immune responses [54]. Indeed, this principle is the very basis of genetic immunization.
• It is necessary to enhance the targeting and specificity of vectors to avoid unpredictable side effects due to the ectopic expression of the transgene in normal tissues. This requirement is essential to generate gene delivery systems suitable for in vivo administration. Most of the human gene therapy protocols currently rely on ex vivo gene transfer manipulations, in which certain cells or tissues must be removed from the patient, transduced in vitro, possibly selected for the expression of the transgene, and then reinfused into the patient. The entire procedure is costly and distressful for the patient. Health care systems and pharmaceutical companies would greatly benefit from the possibility of applying gene therapy approaches based on in vivo gene delivery, as the therapeutic interventions are minimally invasive, and may only require either an injection or the administration of pills [18]. Indeed, the in vivo transduction approach would also allow for a broader application of gene transfer technology in therapy. Certain pathological conditions cannot be dealt with using the ex vivo gene therapy approach, as not all cells or tissues can be surgically removed. Neurons or cardiac cells are an example. However, the in vivo gene therapy approach poses many additional safety concerns versus the ex vivo one. Recent studies have shown there is a possibility that exogenous DNA (transgene and/or viral vector sequences) may eventually be transmitted to the germ line through systematic in vivo administration of viral vectors [55, 56]. Sensitive nested polymerase chain reaction (PCR) techniques have allowed for the detection of low levels of exogenous viral vector DNA in the ovaries and testes of mice, which received systematic administration of adenoviral vectors [56]. Ninety-four percent of these animals tested positive for the presence of adenoviral DNA in the gonads. However, after mating the animals there was no evidence of germ line transmission of adenoviral DNA in the offspring [56]. This issue should also be addressed for in vivo retroviral- or AAV-mediated gene transfer. These viral vectors may have higher probabilities of entering the germ line, as they integrate their chimeric viral genome into host chromosomal DNA [5].
• In many cases, the possibility of regulating transgene expression following cell transduction would be a highly desirable feature. This should allow for the activation of a transgene when it is needed, the maintenance of transgene expression within a therapeutic window, and the possibility of silencing a transgene if necessary. There have been a number of attempts to generate inducible systems. Partial successes have been reported in the in vitro system [5, 57-62] and animal models [63-67]. However, whether transgene regulation can be achieved in patients is still an open question.
• The possibility of combining gene-based interventions with other therapeutics has to be considered.

A broad arsenal of gene transfer systems is currently avalaible [5] and is still in expansion. The characteristics of the main vector systems are described in Table 1. Each gene delivery system has distinct characteristics and preferential applications in therapy [5]. The vectors that have already been applied in clinical trials are based on retroviruses [68-72], adenovirus [73-78], AAV [79-85], vaccinia virus [86, 87], canarypox virus [87], herpes simplex virus (HSV) [88], cationic liposomes [89-92], polylysine-DNA complexes [93, 94], and injection of naked DNA [22, 26, 27, 30]. As anticipated, the pathological conditions with which gene therapy has dealt so far comprise: cancer [2], inherited or acquired monogenic disorders [3, 4], AIDS [3], and cardiovascular diseases [19-21]. In addition, vectors based on vaccinia virus, canarypox virus, injection of naked DNA and other nonviral vectors have been used in the AIDS vaccination programs in the USA [22-24]. Interestingly, viral-based vectors have also been directly administered to patients in order to transduce in vivo cells that are capable of processing the transgene through the antigen-presenting cell pathway. In these cases, the transgene encodes for certain HIV-1 components. The intracellular expression of viral antigens within transduced cells facilitates the cells' antigen-presenting mechanism. In this way various viral epitopes are associated with host HLA class I antigens and expressed on the cell membrane to elicit the host's CTL immune responses [5].

VECTOR SYSTEMS BASED ON RETROVIRUSES, LENTIVIRUSES AND FOAMI VIRUS Retroviruses have attracted a great deal of interest from the standpoint of gene transfer applications [5]. Such interest is certainly motivated by the characteristics of the biology of retroviruses, which belong to the genera of the retroviridae. This category also comprises lentiviruses and foami viruses. The retroviridae have a long history of cross-species infections [116, 117]. They have been responsible for many zoonotic events (transmission of infectious agents from animals to humans) [116] which indicates that they may be suitable for DNA delivery into humans. The retroviral genome is relatively simple [118], so it may easily be rearranged to generate recombinant viral vector particles which are noncompetent for replication [5], and which can sustain only one round of infection. Retroviral vectors are mainly based on the amphotropic Moloney murine leukemia virus (MLV) [118], and have been used in many gene therapy clinical trials for the treatment of cancer [2, 5], inherited or acquired monogenic disorders [5], and AIDS [119-124]. Lentiviral vectors are based on HIV-1 [98-104] or on FIV [105-107]. Neither lentiviral- or foami virus-based vectors have been used in clinical trials yet. However, the HIV-1-based lentiviral vector system is unlikely to be approved for clinical trials for a variety of reasons. First is the issue of the serum conversion of the patients to HIV-1. Secondly, is the production and administration of lentiviral vector stocks require category three facilities. Third, the large quantities of lentiviral vector stocks that have to be produced for the clinical trials pose an additional concern in the matter of biosafety. Fourth, this vector system is already obsolete, due to the development of the FIV-based lentiviral vector system, which has circumvented all the above-mentioned issues. In fact, FIV has been certified for category two manipulations, and is based on a lentivirus which cannot infect humans. Therefore, the serum conversion to FIV does not raise any concern. The characteristics of the retroviridae vector systems are summarized in Table 1. All these viral vector systems can be produced at relatively high titers (106-107 cfu/ml) [5]. A property of retroviruses is that they can only infect dividing cells, as they need the breakdown of the nuclear membrane to be able to deliver the preintegration complex into the cell nucleus [125]. Conversely, lentiviruses [98-107] and, to a lesser extent, foami viruses [95-97, 126] can also infect nondividing cells. The requirement for active cell division can be either an advantage or a drawback for retroviral vectors. The selective transduction of dividing cells makes retroviral vectors suitable for cancer therapy [5]. On the other hand, retroviral vectors cannot be used for a variety of therapeutic applications, such as neurologic diseases and a number of genetic diseases that require the transduction of hepatocytes [127], as neurons and hepatocytes do not divide. In all these respects, FIV-based lentiviral vectors may find useful applications. Indeed, retroviral vectors have been used in a number of preclinical studies for liver-directed gene transfer and in some clinical trials [127]. The procedure used was based on ex vivo or in vivo transduction of hepatocytes, which were induced to proliferate by complex and artificial procedures [127]. Retroviral-mediated ex vivo transduction relies on stimulating cell division by culturing primary hepatocytes in appropriate media [127]. This approach has been employed in preclinical studies for the following genetic diseases: type I tyrosinemia, familial hypercholesterolemia and α1-antitripsin deficiency [127]. One clinical trial was conducted to treat familial hypercholesterolemia by retroviral-mediated ex vivo gene transfer. The low-density lipoprotein (LDL) receptor gene was introduced into hepatocytes that had been surgically removed from patients, and which were then reinfused into the liver following gene transduction [128, 129]. The procedure was safe but there was no convincing evidence of therapeutic efficacy [127]. Liver biopsies were removed after treatment, and few cells tested positive for the expression of LDL-receptor [127], indicating that the transduction efficiency was not high, or that transduced cells were lost or eliminated after reinfusion into the liver. In vivo retroviral-mediated transduction of hepatocytes is even more complicated, as it requires artificial regeneration of the liver [127]. This may be achieved by a variety of means: partial hepatectomy, chemical injury, administration of growth-stimulating drugs or vascular occlusion [127]. Experiments in animal models have shown efficient retroviral-mediated gene transfer into the liver of rodents [127], but a poor efficacy of intervention in larger animals such as dogs [127]. This is probably due to the different kinetics of liver regeneration between large mammals and rodents. In conclusion, in vivo administration of retroviral vectors into the liver does not seem applicable to humans. Probably, the development of a retroviral vector system based on the hepatitis B virus may facilitate liver-directed gene delivery. In this respect, a hepatitis B-based retroviral vector is under development [130, 131]. Interestingly, one study has shown successful liver-directed hepatitis B viral-mediated gene transfer of green fluorescence protein. In addition, the delivery of type I interferon by hepatitis B-based retroviral vector has suppressed endogenous wild-type virus replication in the duck model of hepatitis B virus infection [131]. However, this viral vector system needs further characterization, and should also be adapted to the rodent animal model before considering its application in clinical trials. All the viral vectors based on retroviridae can be used to transduce a wide range of cell types. This is due to the fact that HIV-1, FIV and foami virus cores can be pseudotyped with the MLV amphotropic envelope or vesicular stomatitis virus G (VSV G) glycoprotein (Table 1) [132, 133]. Pseudotyping with the VSV G glycoprotein also allows for easy purification of the various viral vector particles, as they became more stable and resistant, so they can be isolated from the cell culture supernatants by simple ultracentrifugation [134]. Foami viral vectors have a broad cell tropism, even without being pseudotyped with MLV amphotropic envelopes or with VSV G glycoprotein [95-97,126]. Interestingly, wild-type foami viruses are resistant to complement-mediated lysis [95] and have a total insert capacity in the virion of approximately 14kb [95]. Conversely, MLV-based retroviral, lentiviral and foami viral vectors pseudotyped either with amphotropic retroviral envelopes or VSV G glycoprotein are susceptible to complement-mediated lysis [135-138] and their total insert capacity in the virion is in the range of 10kb [5]. It has been demonstrated that packaging cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars generate enveloped viruses that are more susceptible to complement attachment [136]. The viral systems analyzed in this study were based on VSV, HIV-2 and human foami virus [136]. It has been argued that the humoral immune response to alphaGal may be a mechanism of defense against the transmission of viral agents from animals to humans [136], and that viral vectors for human gene therapy should be produced from alphaGal-negative cells [136]. Another study has reported the production of MLV-based amphotropic retroviral vectors resistant to human complement [139]. This was achieved by expressing hybrid amphotropic envelopes on the viral membrane. These hybrid amphotropic envelopes were generated by fusing in frame the catalytic domain of the human complement regulatory protein decay-accelerating factor with a portion of the envelope [139]. The possibility of concentrating retroviral, lentiviral, and foami viral vector particles may improve the transduction efficiency for both ex vivo and in vivo applications. The protection from complement-mediated lysis is particularly required for the optimization of in vivo gene transfer models. A number of other studies have been conducted to further improve the performance of retroviral vectors in preclinical studies and clinical trials. A simple approach consists of using enhanced green fluorescence protein as reporter gene [140-143]. This allows for the rapid detection and isolation of the fraction of cells that have been transduced ex vivo. In addition, the green fluorescence protein can be readily detected in tissues following infusion of transduced cells into the animals [140, 141]. Other strategies to improve the retroviral transduction efficiency are based on the artificial induction of cell division. This can be achieved in many ways: preincubation of primary cultures of hematopoietic stem cells with various interleukins (IL-2, IL-3, IL-6) and/or other growth factors or colony-stimulating factors [144-146]; combination of retroviral- and lipofectAMINE-mediated gene transfer into stem cells prestimulated with IL-2 (in this study, lipofectAMINE was used to facilitate the delivery of retroviral vectors into the target cells) [147]; colocalization of retroviral particles and hematopoietic stem cells on specific fibronectin fragments (Retronectin) [148]; combination of Retronectin system with prestimulation of hematopoietic stem cells with ILs or other growth factors [149, 150]. Ex vivo retroviral transduction of human hematopoietic stem cells also has several disadvantages. Besides being costly and time-consuming, this approach may introduce some artifacts into hematopoietic stem cells. For instance, the in vitro culture conditions may impair the ability of transduced hematopoietic stem cells to engraft once they are reinfused into the subject. This situation has already been mentioned for the gene-based clinical trial for the treatment of familial hypercholesterolemia, in which the target cells were hepatocytes [127]. The tissue culture conditions for the ex vivo propagation and transduction of human hematopoietic stem cells are conducted at nonphysiologic cell concentrations, and require the combination of growth factors that may induce cell differentiation and, therefore, pose a limitation to the long-term engraftment of the transduced cells. It has been observed that HIV-1- and FIV-based lentiviral vectors may be more suitable for the transduction of hematopoietic cells than amphotropic retroviral vectors [98, 100, 104, 107]. The ability of lentiviruses to also infect nondividing cells may circumvent the issue of prestimulating hematopoietic stem cells [151]. Moreover, lentiviruses usually yield higher transduction efficiency of primary stem cell cultures than retroviral vectors [152, 153]. However, an important aspect that must be addressed in the matter of lentiviral-mediated gene transfer is to establish whether the transfer vector remains episomal in the nucleus of transduced cells that are in G0 phase. Transgene expression detected following lentiviral transduction of quiescent cells may indeed derive from extrachromosomal double-stranded DNA transfer vector. If this is the situation, lentiviral transduction of quiescent cells may only allow for transient expression of the transgene. An important safety issue in the matter of viral-mediated gene transfer is the formation of viral-competent viruses in patients, which may occur by homologous recombination events within the packaging cell lines. Retroviral vector stocks are routinely monitored in clinical trials for the absence of replication-competent retroviruses (RCR) [154]. The techniques are essentially based on sensitive PCR and serological enzyme-linked immunosorbent assay [154]. In addition, retroviral stocks must be tested for the absence of endotoxins and various contaminating agents, such as bacteria and fungi, which may be acquired during the propagation of packaging cell lines or target cells [119, 155]. The purity of the various genetic material used in the trial must also be tested [119, 155]. The RCR formation is a rather unlikely event due to the design of retroviral vector. The current trend is to produce high titer retroviral vector stocks transiently [5] in order to further minimize the possibility of recombination events among the various retroviral components in the packaging cell line. These transient systems are based on three plasmid cotransfections of the highly transfectable 293T cell line [156]. As reviewed elsewhere [5], the proviral genome has been broken down into three parts, and overlapping sequences have been mostly removed. The RCR formation is unlikely due to the fact that it would require simultaneous rearrangement among three different plasmids in a specific configuration in a very limited period of time. The transfection procedure usually takes between 48 to 72 h to produce the retroviral vector stocks [5]. So far, the retroviral vectors used in clinical trials derive from conventional packaging cell lines, which were previously approved for clinical applications by the U.S. Food and Drug Administration [3, 157]. Studies are currently addressing the issue of generating clinical grade retroviral vector stocks by transient transfection systems [158]. Another safety concern is the possible recombination between retroviral vectors and HERVs in patients (Table 1). The human genome contains thousands of HERV sequences [159-161], most of which are defective genes. These HERV sequences derive from ancient retroviral infections [160] in which transmission occurred either in germ line cells or cells in the early embryo [160, 161]. About 1% of the human genome is composed of HERV-related sequences [161], and probably more than 10% of the human genome may have evolved through reverse transcription mechanism [161]. So far, only one HERV has been found that encodes for a complete viral particle, which was named HERV-K [162]. However, HERV-K is not competent for replication [162]. The biological relevance of HERVs deserves further investigation. HERVs have some possible advantageous effects in fundamental biological processes such as: development and/or differentiation, protection from superinfection by exogenous retroviruses, protection of the embryo from retroviral infection (germ line vaccination), cell fusion, tissue-specific gene expression, alternative splicing, and polyadenylation [161]. The potential pathogenicity of HERVs cannot be predicted. They may be involved in the development of malignancies and autoimmune diseases [161]. The envelope of an HERV may either protect the host from exogenous retroviral infection in a receptor interference fashion [163] or dysregulate the local cellular immunity through a superantigen-encoded region, as proposed for type I diabetes [164]. A study has observed that the multiple sclerosis-associated retrovirus detected in the plasma of patients with multiple sclerosis [165, 166] has high homology to an HERV [167], which was named HERV-W. Xenotransplantation techniques and gene therapy approaches based on retroviridae vectors may eventually tamper with the biology of HERVs [161]. Retroviral vectors may recombine with HERVs in patients, and generate a variety of possible adverse effects. At this point in time, we cannot predict possible adverse effects of recombination due to the lack of sufficient information about HERVs. What one can expect is the formation of RCR in patients, or the expression of HERV genes that were silent prior to gene therapy or xenotransplantation intervention. If such events should occur, most likely the subject may develop cancer or become susceptible to immune system dysregulation. The integration of the retroviral genome into chromosomes allows for stable transgene expression. This stability is also due to the low degree of retroviral particle immunogenicity. This is in contrast with what has been observed for adenoviral-mediated gene transfer, where transgene expression is only transient. There are two reasons for the transient nature of transgene expression in adenoviral-mediated gene transfer. First, the adenoviral genome does not integrate into the host chromosomal DNA [5]. Second, the adenoviral particles are immunogenic [5] and express leaky adenoviral genes that render the transduced cells susceptible to CTL immune responses [168-171]. Stable retroviral-mediated transgene expression is desirable for the treatment of diseases that require long-term expression of the transgene, such as genetic disorders and neurologic illnesses [5]. However, the duration of transgene expression is still not optimal. This is because the retroviral long terminal repeats (LTRs) are susceptible to methylation in CpG-rich islands, which may silence the gene transcription [172, 173]. The incidence of this phenomenon depends on the type of transduced cells and the site of retroviral genome integration [174]. It has been shown that Sp1 binding sites may, to some extent, prevent the methylation of the promoter [175]. Retroviral vectors based on murine embryonic stem cell virus (MESV) [176, 177] and on murine stem cell virus (MSCV) [178] have been engineered to optimize the duration of transgene expression in undifferentiated murine embryonic and hematopoietic cells [176-178]. To this end, the LTRs of the MESV- and of the MSCV-based vectors have been modified. In the MESV vectors, the 5′-LTR contains an extra Sp1 binding site, which has been introduced by a point mutation [176, 177]. This has optimized, to some extent, the duration of transgene expression in embryonic and hematopoietic cells. However, silencing of transcription has been observed following the differentiation of embryonic stem cells [179]. The MSCV-based vectors, in addition to the point mutation that creates an Sp1 binding site, contain another point mutation that destroys the binding site of the embryonal LTR-binding protein (ELP) [178]. ELP is a transcriptional suppressor of the activity of the MLV 5′-LTR in undifferentiated murine embryonal carcinoma cells [180]. These modifications have further improved the performance of retroviral vectors in terms of duration of transgene expression. However, better evaluation of the exact extent of this improvement in in vivo systems is needed. The random insertion of the retroviral transfer vector has several drawbacks: it may damage the cell genome, cause the inactivation of tumor suppressor genes, or induce the expression of cellular oncogenes. Probably, this is not sufficient to generate a neoplasia, as cancer is a multistep process which requires a combination of genetic alterations and the expression of cellular and/or exogenous oncogenic factors [181]. However, if the transduced cells should be genetically impaired by the random insertion of the viral vector's genome, this would at least predispose the cells to undergo to neoplastic transformation. To date, human gene therapy protocols have been applied only to a limited number of patients, and most of them did not have a long life expectancy. An important question is what happens if retroviral-mediated gene transfer is applied to larger scale clinical trials and subjects who have a life expectancy in the range of some decades? The current development of preventive cancer prognosis cannot answer this question, so it is not possible to properly assess the ratio of benefit to risk for all the patients. A recent study has addressed the issue of cell transformation induced by retroviral-mediated gene transfer in an in vitro system [182]. Mouse fibroblasts BALB/c-3T3 cells were transduced with a retroviral vector, and the transformation frequency was compared to that of the untransduced cells [182]. The parental cell line undergoes spontaneous transformation that is in the range of 1.1 × 10–5 [183]. In this study, the transformation rate of retrovirally transduced BALB/c-3T3 cells was in the same range [182]. The number of integrated proviral copies per cell genome varied from one to six, depending on transduction efficiency [182]. So, improved transduction efficiency is correlated with better transgene expression, which in turn is due to the higher number of integrated retroviral transfer vector's copies per cell genome. But this is also proportional to the higher risk of mutagenic events. Previous studies on retroviral-induced mutagenesis in mammalian cells have found a ratio of “mutations versus insertional events” which ranged from 10–9 to 10–3 [184-189]. Such variability indicates that the ratio of mutations per insertional events depends on the cell type and assay system. This ratio should be established for human primary lymphocytes, which normally are not retrovirally transduced as efficiently as mouse fibroblasts [182, 190, 191] or other cultured cell lines [191]. However, the lower transduction efficiency, per se, does not guarantee a lower ratio of “mutations versus insertional events” in human primary lymphocytes. All these findings suggest that in the design of clinical protocols using retroviral-mediated gene transfer, the average number of integrated viral genomes should be carefully evaluated. Such a procedure is feasible for ex vivo retroviral-mediated gene transfer, but not for the in vivo administration system. Overall, the in vivo administration of retroviral vectors poses a number of additional safety concerns and technical limitations if compared to the ex vivo gene transfer model. To pursue the goal of safe and efficient in vivo retroviral transduction, it is necessary to generate tissue- or cell-specific retroviral vectors, which can integrate their genome in safe cell chromosomal sites. The latter issue has never been tackled, whereas the engineering of ecotropic-based retroviral vectors with altered cell tropism has attracted much attention [5], but all the attempts had little success. The chimeric retroviral particles that have been produced have a low transduction capacity [5], or even fail the gene transfer process [192]. To date, the ex vivo retroviral-mediated gene transfer model is more realistic than the in vivo one, although it is not optimal for gene therapy applications. Also, from the standpoint of safety concern, the ex vivo procedure can be more easily monitored.