Lentiviral Vectors for Gene Therapy PDF

46 Fig. 3.6 Different lentiviral vector generations used in gene therapy and their modifications engineered from the wild-type HIV-1. The first generation of lentiviral vectors was produced using three plasmids encoding the viral proteins. In these vectors, only the transgene contained the essentia...

46 Fig. 3.6 Different lentiviral vector generations used in gene therapy and their modifications engineered from the wild-type HIV-1. The first generation of lentiviral vectors was produced using three plasmids encoding the viral proteins. In these vectors, only the transgene contained the essential cis-acting elements (LTRs, Ψ, RRE) that allowed their packaging in the produced lentiviral particles. In the second generation of lentiviral vectors, the number of plasmids was maintained, but in order to improve their safety the accessory genes were deleted. In the third gen- 3 Viral Vectors for Gene Therapy eration, the tat gene was eliminated and the viral genome divided into an additional plasmid containing the rev gene. This also included the deletion of the U3 region of the 3’LTR, generating self-inactivating virus. Finally, a fourth generation of lentiviral vectors was developed to further reduce the risk of recombination between the plasmids used. For that, the segments gag and pol were codon optimized. In this fourth generation, all the modifications of the previous generations were maintained. 3.1 Lentiviral Vectors 47 Table 3.4 Main features of the different generations of lentiviral vectors Constructs Packaging plasmids Accessory genes tat and rev genes gag and pol genes 3’LTR deletion Codon optimization First generation 3 1 Second generation 3 1 Yes Same plasmid Same plasmid No No No Same plasmid Third generation 4 2 Fourth generation 4 2 Same plasmid No tat absent, rev in a separate plasmid Same plasmid No tat absent, rev in a separate plasmid Same plasmid No No Yes No Yes gag and pol genes Table 3.5 Main advantages and disadvantages of third generation lentiviral vectors. Advantages Able to transduce slowly dividing and nondividing cells Delivered transgenes are more resistant to transcriptional silencing Suitable for several ubiquitous or tissue-­ specific promoters Safety increased by self-inactivation Disadvantages Production of high titers is more difficult Still some possibility of generating replication-­ competent lentiviruses megalovirus) promoter. The addition of a new plasmid, separating the rev gene, further increased the safety of the viral vectors production by reducing the possibility of RCL generation. An additional improvement was also added to these lentiviral vectors with the deletion of part of the U3 region in the 3’LTR, generating SIN (self-inactivating) vectors, thus decreasing the risk of activating nearby genes in the integration process. These lentiviral vectors have only three of the nine wild-type HIV-1 genes, which significantly increases their safety profile. The four constructs used for their production are (i) a packaging plasmid with the gag and pol genes; (ii) a plasmid with rev gene, (iii) a plasmid with the env (or VSV-G or other pseudotyped protein), and (iv) a plasmid with the transgene (and a strong heterologous promoter). Fourth Generation The potential formation of RCLs using the third generation lentiviral vectors is very reduced, however, theoretically, it is still possible that homologous recombination between the transfer and the packaging plasmids takes place. To solve this problem, codon optimization was implemented in the gag and pol genes, thus eliminating the homology between plasmids. This fourth generation of lentiviral vectors displays improved biosafety; however, viral titers were negatively affected. Maybe because of this, it has not been extensively used, and the previous lentiviral vector generations are still more commonly applied, especially the third generation due to its important advantages (Table 3.5). 3.1.3 Additional Improvements to Lentiviral Vectors Several other improvements in lentiviral vector design were introduced in the different generations to improve efficiency, to facilitate their production or, as already mentioned, to improve their biosafety (Fig. 3.7) [12]. One of those improvements was the introduction of a cis-­ acting polypurine tract (cPPT), to increase the viral vector transduction efficiency both in vitro and in vivo. Another strategy, improving lentiviral vector expression, was the introduction of the woodchuck hepatitis virus post-­ 48 3 Viral Vectors for Gene Therapy Fig. 3.7 Modifications introduced to lentiviral vectors relative to the wild-type HIV-1 virus, in order to improve their safety profile while maintaining their efficiency and production in high titers. transcriptional regulatory element (WPRE), which increases the number of unspliced RNA molecules, leading to an increase of the transgene expression in target cells. One important issue resulting from lentiviral vector-mediated integration is the possibility of transgene expression repression, due, for example, to epigenetic events. To address this problem, chromatin-insulator sequences were introduced in the lentiviral vectors. Insulators have the ability to block enhancer-promoter interactions and/or serve as barriers against silencing effects. The further engineering of lentiviral vectors also included the fusion of proteins or antibodies to envelope glycoproteins to alter their tropism and retarget the vectors to specific cells. Another commonly used strategy to direct and specify transgene expression is to include a tissue-specific promoter in the transgene construct. This strategy will not select the cells to transduce, but rather limit the expression of the transgene to a certain cell type due to the presence of the specific promoter. Limiting expression of the transgene to particular cells is particularly useful in the context of the central nervous system, where the presence of different cells types makes specific transduction difficult. SIN Vector Design Another feature included in most of the lentiviral vectors is the inclusion of a self-inactivating long terminal repeat (SIN-LTR), in which the U3 3.1 Lentiviral Vectors Fig. 3.8 Self-inactivating lentiviral vectors. In wild-­ type HIV-1 virus, the viral RNA is transcribed under the control of an enhancer-promoter sequence that is located in the U3 region of the LTRs, being required for the production of new viral particles and the continuation of the replicative cycle. During the process of reverse transcription 49 the U3 region of the 3’LTR is transposed to the 5’LTR. In self-inactivating vectors, the U3 region of the 3’LTR is partially deleted in order to block its enhancer-­promoter function. Upon integration, the proviral DNA will harbor the partially deleted U3 region in both LTRs, thus preventing the further continuation of the replicative cycle. region in the 3’LTR has been partially deleted deficient), this region is dispensable, as the pack(Fig. 3.8) [13]. This feature reduces the risk of aged RNA expression is driven by the 5’LTR lentiviral vector recombination with wild-type region and the transgene expression by the respecviruses, thus increasing its safety. In the wild-­ tive promoter. The partially deleted U3 region type HIV-1, the U3 region acts as a viral enhancer/ will be transfered to both the 5’ and 3’LTR promoter, being essential for viral replication, regions during integration, so that any viral parespecially for the formation of both the 5’ and ticle progeny that is formed will harbor two 3’LTR of new viral particles. However, in the ­inactivated LTRs and transgene expression will recombinant lentiviral vectors (replicative-­ be only driven by the internal promoter. 3 50 Non-integrative Lentiviral Vectors In the context of gene therapy, one of the main advantages of using lentiviral vectors is their ability to stably integrate the desired transgene in the target cells genome. However, integration can lead to the development of undesired consequences, such as the activation of oncogenes. In fact, for some applications the development of non-integrative vectors would still be effective, having as an additional benefit a better safety profile [14]. To attain this goal, lentiviral vectors were designed containing mutations in regions that are essential for the proviral DNA integration process, namely in, (i) the U3 region of the 5’LTR, (ii) the U5 region of the 3’LTR, and (iii) the integrase protein itself. Lentiviral Vector Pseudotyping As mentioned, the inclusion of different glycoproteins into the envelope of lentiviral vectors alters and increases the variety of target cells that can be transduced, thus improving their tropism. Like it was referred, pseudotyping is the process of producing viral vectors (or virus) containing envelope proteins from a different virus [15]. Several glycoproteins from different virus have been used and studied to pseudotype lentiviral vectors providing different advantages, such as increasing their efficiency or reducing their toxicity (Table 3.6). 3.1.4 Lentiviral Vector Production Any delivery system used for gene transfer, either non-viral or viral, has to be produced/built preferentially with a safe and simple method of low technical complexity that, of course, yields the system in high amounts. In the case of lentiviral vectors, the safety issue was a major driving force in the development of production methods and the main reason why the viral genome was separated into different constructs. However, this important advance brought a new challenge, as all the constructs must join together in the factory cells to produce effective lentiviral vector particles. To accomplish this, two transfection methods are used: transient transfection [16] and Viral Vectors for Gene Therapy Table 3.6 Common viral glycoproteins used for lentiviral vector pseudotyping. Species/envelope Vesicular stomatitis virus (VSV-G) Feline endogenous retrovirus (RD114) Ebola Rabies Lymphocytic choriomeningitis virus (LCMV) Ross River virus Influenza virus hemagglutinin Moloney murine leukemia virus 4070 envelope Advantages Wide tropism Facilitates production using ultracentrifugation More efficient and less toxic than VSV-G in hematopoietic cells Efficiently transduces airway epithelium Retrograde transport in neuronal axons Low toxicity Transduces hepatocytes, glial cells, and neurons Transduces airway epithelium Able to transduce most cells stable transfection [11]. In the first approach, all the plasmids (three or four, depending on the generation used) are simultaneously transiently transfected into a packaging cell line (Fig. 3.9). Several transfection agents cab be used, like calcium phosphate, polyethylenimine (PEI) or cationic lipids. The second approach involves the use of stable packaging cell lines already containing one or more viral genes, thus reducing the number of plasmids to be transfected [4]. Both approaches have advantages and drawbacks, and their choice is mainly dependent on the laboratory conditions, on the safety regulations and on the viral titer needed. Other important issues to be considered in the production of lentiviral vectors include the method of viral particle purification and concentration, the procedure for titer assessment, the choice of producer cell line and the production scale (Fig. 3.10). For research purposes, the production of lentiviral vectors is well-established in human embryonic cells kidney 293 (HEK293) or HEK293T adherent cells using transient transfection methods. However, for clinical trials, the quantity and quality of the vectors needed are 3.1 Lentiviral Vectors Fig. 3.9 Plasmids used to produce lentiviral vectors in different vector generations. Depending on the generation, lentiviral vectors can be produced by transfecting three or four plasmids into producing cells. Alternatively, 51 a stable cell line expressing one or two viral genes could be used in the production, thus reducing the number of plasmids to be transfected. 52 3 Viral Vectors for Gene Therapy Fig. 3.10 Overview of the main steps of lentiviral vector production. Several methods can be used to transfect viral plasmids, including delivery mediated by liposomes. The produced viral particles can be concentrated and purified from the producing cells using different techniques, among which ultracentrifugation is the most common. Finally, several methods to quantify the lentiviral vectors titer can be used, although the quantification of p24 is the most common. much higher. Thus, large-­ scale methods were developed using both adherent and suspension cell cultures. The process is quite expensive and complex, and only a few sites in Europe and the USA are able to produce GMP lentiviral vector particles in large scale [11]. Concentration and purification methods are both very important in the production of lentiviral vectors and are closely related to the production scale needed (research or clinical trial). Even though, probably the method most commonly used for concentration is the centrifugation of culture medium after an initial filtration. This method can also be combined with anion-­ exchange chromatography for an additional purification step. However, these methods are not suitable for large-scale productions and in vivo application, as often they are not free from culture medium/process contaminants. Therefore, in large-scale productions, several methods are combined to achieve three main goals: (i) an initial purification of the vectors; (ii) an intermediate purification to remove specific impurities, and (iii) final refining to remove trace contaminants. Finally, the last step in the production of lentiviral vectors, which is the assessment of the viral production titer, is also very important, in order to adjust vector doses and to evaluate the transduction efficiency. There is a wide range of methods used for this purpose, for example (i) measuring the quantity of one vector component (commonly the viral p24 protein is used); (ii) measuring the number of provirus DNA copies in infected cells; or (iii) measuring transgene or reporter gene expression in infected cells. 3.2 Gamma Retrovirus 53 Several modifications and improvements are continuously being tested aiming to increase lentiviral vector quality and quantity and also to increase safety in the production process. 3.1.5 Lentiviral Vectors in Clinical Trials There is already one gene therapy product approved in Europe and the USA based on the delivery by lentiviral vectors. Kymriah® is an ex vivo gene therapy delivering CD19-specific CAR T-cells, indicated for the treatment of acute lymphoblastic leukemia. Due to their important advantages, lentiviral vectors were and are used with success in several clinical trials, particularly for rare diseases. More recently, they were also applied in gene therapy studies for more frequent genetic and acquired diseases, including β-thalassemia, Parkinson’s disease and cancer. Tables 3.7 and 3.8 detail two examples of gene therapy clinical trials using lentiviral vectors [17, 18]. 3.2 Gamma Retrovirus Gamma retrovirus also belongs to the Retroviridae family (retroviruses), like the lentivirus. However, their genome organization is simpler [19], which makes its genetic engineering easier than for lentivirus (Fig. 3.11). As vectors for gene therapy, they share more or less the same advantages and disadvantages of the lentivirus. However, contrary to those, the gamma retrovirus only infects dividing cells, and maybe for that, currently, they are less used in gene therapy than lentivirus [20]. Moreover, lentiviral vectors seem to be safer than gamma retrovirus in relation to the risk of insertional mutagenesis. The gamma retrovirus was mainly used in the earlier gene therapy studies, until the previously mentioned incident with a X-linked severe combined immunodeficiency (SCID) trial (Table 3.9) [21] where insertional oncogenesis was observed in some of the trial subjects. Table 3.7 Example 1 of a gene therapy clinical trial using lentiviral vectors as the delivery system of the therapeutic gene. Study Disease Therapeutic gene Delivery vector Clinical trial Inclusion criteria Type of administration Clinical outcome Palfi, S., et al. (2014) Long-term safety and tolerability of ProSavin, a lentiviral vectorbased gene therapy for Parkinson’s disease: a dose escalation, open-label, phase 1/2 trial, Lancet 383, 1138–1146 Parkinson’s disease, which is a common neurodegenerative disease mainly characterized by motor impairments, resulting from the progressive degeneration of dopaminergic neurons in the substantia nigra that project axons to the striatum, where dopamine is released. ProSavin (genes encoding key enzymes in the dopamine metabolism, tyrosine hydroxylase, AADC, and cyclohydrolase 1) in three doses (low 1.9 × 107 TU; mid 4.0 × 107; and high 1 × 108) Lentiviral vector (based on equine infectious anemia virus) produced by triple transient transfection methods in HEK293T cells, purified and concentrated by anion-exchange chromatography and hollow fiber ultrafiltration Phase I/II (long-term safety and tolerability), open-label and 12-month follow-up study, in France and the UK 48–65 years; disease manifestation for 5 or more years and 50% or higher motor response to oral dopamine treatment Local delivery of ProSavin bilaterally into the striatum Despite some adverse events, the study found that ProSavin was safe and well tolerated. No detectable antibody response against any of the ProSavin transgene products was detected. There was an improvement in motor behavior in all the treated patients, and 11 of the patients had a reduction in the daily levodopa administration at 6 and 12 months after the therapy.