Packaging cell lines for retroviral vector manufacture (1 – RMCE – Recombinase Mediated Cassette exchange; NR – Not reported: the titers reported for these packaging cells are expressed in terms of reverse transcriptase activity, which the correlation with infectious titers depends on the cell system.)
Gamma-retroviral vectors, commonly designated retroviral vectors, were the first viral vector employed in Gene Therapy clinical trials in 1990 and are still one of the most used. More recently, the interest in lentiviral vectors, derived from complex retroviruses such as the human immunodeficiency virus (HIV), has been growing due to their ability to transduce non-dividing cells (Lewis et al. 1992; Naldini et al. 1996), an attribute that distinguishes them from other viral vectors, including their simple counterparts, gamma-retroviral vectors. Retroviral and lentiviral vectors most attractive features as gene transfer tools include the capacity for large genetic payload (up to 9 kb), minimal patient immune response, high transducing efficiency
According to the most recent updates, retroviral and lentiviral vectors represent 23% of all the vector types and 33% of the viral vectors used in Gene Therapy clinical trials. Moreover, retroviral vectors are currently the blockbuster vectors for the treatment of monogenic and infectious diseases and gene marking clinical trials (Edelstein 2010).
Retroviruses are double stranded RNA enveloped viruses mainly characterized by the ability to “reverse-transcribe” their genome from RNA to DNA. Virions measure 100-120 nm in diameter and contain a dimeric genome of identical positive RNA strands complexed with the nucleocapsid (NC) proteins. The genome is enclosed in a proteic capsid (CA) that also contains enzymatic proteins, namely the reverse transcriptase (RT), the integrase (IN) and proteases (PR), required for viral infection. The matrix proteins (MA) form a layer outside the capsid core that interacts with the envelope, a lipid bilayer derived from the host cellular membrane, which surrounds the viral core particle (Coffin et al. 1997). Anchored on this bilayer, are the viral envelope glycoproteins (Env) responsible for recognizing specific receptors on the host cell and initiating the infection process. Envelope proteins are formed by two subunits, the transmembrane (TM) that anchors the protein into the lipid membrane and the surface (SU) which binds to the cellular receptors (Fig. 1).
Based on the genome structure, retroviruses are classified into simple (e.g. MLV, murine leukemia virus) or complex retroviruses (e.g. HIV) (Coffin et al. 1997). Both encode four genes:
2. Cell line platforms for the production
The establishment of retroviral and lentiviral producer cells, named packaging cell lines, has been based on the physical separation of the viral genome into different transcriptional units to minimize the risk of generating replication-competent particles (RCPs) (Fig. 3). Some of
these constructs are additionally engineered with heterologous sequences including: promoters (Dull et al. 1998) to support their independent expression or for improved safety, enhancers (Gruh et al. 2008) and stabilizing elements (Zufferey et al. 1999) to increase the overall levels of transcripts both in producer and target cells, hence increasing viral titers and transgene expression.
2.1. Retroviral vectors
For both retroviral and lentiviral vector production, different packaging systems, named generations, have been developed. Each new generation aimed at minimizing and reduce the risk of RCPs formation face to the previous one (Fig 3).
In the case of vectors based on MLV or other simple retrovirus, the non-cytotoxicity of the viral genes has allowed the establishment of cell lines stably and constitutively expressing viral vectors. Table 1 lists some of the available retroviral vector packaging cell lines.
The first packaging cells reported as so for simple retroviral vector production were established by providing the packaging functions (
|Ψ-AM||Murine NIH 3T3||Amphotropic||2.0 x 105||MLV based||1st||(Cone and Mulligan 1984)|
|PA317||Murine NIH 3T3||Amphotropic||3.0 x 106||MLV based||2nd||(Miller and Buttimore 1986)|
|Ψ-CRIP||Murine NIH 3T3||Amphotropic||6.0 x 106||MLV based||3rd||(Danos and Mulligan 1988)|
|PG13||Murine NIH 3T3||GaLV||5.0 x 106||MLV based||(Miller et al. 1991)|
|Gp + envAm12||Murine NIH 3T3||Amphotropic||1.0 x 106||MLV based||(Markowitz et al. 1988)|
|HAII||Human HT1080||Amphotropic||1.0 x 107||MLV based||(Sheridan et al. 2000)|
|FLY A4||Human HT1080||Amphotropic||1.0 x 107||MLV based||(Cosset et al. 1995)|
|FLY RD18||Human HT1080||RD114||1.2 x 105||MLV based||(Cosset et al. 1995)|
|Te Fly A||Human Te671||Amphotropic||1.0 x 107||MLV based||(Cosset et al. 1995)|
|Te Fly Ga 18||Human Te671||GaLV||1.0 x 106||MLV based||(Cosset et al. 1995)|
|CEM FLY||Human CEM||Amphotropic||1.0 x 107||MLV based||(Pizzato et al. 2001)|
|293-SPA||Human 293||Amphotropic||6.0 x 106||MLV based||(Davis et al. 1997)|
|293||Human 293||Amphotropic Xenotropic 10A1||NR||MLV based||(Farson et al. 1999)|
|Phoenix||Human 293T||Amphotropic||1.0 x 105||MLV based||(Swift et al. 2001)|
|Flp293||Human 293||Amphotropic||2.0 x 107||MLV based||3rd with RMCE1 technology||(Schucht et al. 2006)|
|293 FLEX||Human 293||GaLV||3.0 x 106||MLV based||(Coroadinha et al. 2006b)|
|PG368||Murine NIH 3T3||GaLV||1.0 x 106||MLV based||(Loew et al. 2009)|
Retroviral vectors have been based on several viruses including avian, simian, feline and murine retroviruses, being the latter (MLV) the most used. As so, the majority of the retroviral vector packaging cell lines established were murine derived, being NIH/3T3 the most widely employed. However, it was rapidly found that the presence of galactosyl(α1-3)galactose carbohydrate moieties produced by murine cells in retroviral envelope lead to its rapid detection and inactivation by the human complement system (Takeuchi et al. 1994; Takeuchi et al. 1997; Takeuchi et al. 1996). Nowadays, murine cells are being replaced by human cell lines, to reduce the possibility of endogenous retroviral sequences packaging and also to improve vector half-life
Establishing a producer cell line involves at least three transfection and clonal selection steps, taking a time-frame of around one year which constitutes a major drawback in stable cell line development (see section 3.1). Yet, this process is undertaken for each new therapeutic gene and/or different envelope protein required (for changing vector tropism). On the other hand, high-titer packaging cells development has been based on an efficient method to facilitate the selection of a high producer cell clone in which a selectable marker gene is inserted in the vector construct downstream of the viral genes, so they are translated from the same transcript after ribosomal reinitiation (Cosset et al. 1995). This strategy, however, although very efficient for screening stable integration and/or high level long-term viral genome expression, raises considerable problems in therapeutic settings including immune response against the selection (foreign) gene product(s) (Liberatore et al. 1999). Therefore, a new generation of retrovirus packaging cell lines based on cassette exchange systems that allow for flexible switch of the transgene and/or envelope, as well as selectable marker(s) excision, were developed (Coroadinha et al. 2006b; Loew et al. 2004; Persons et al. 1998; Schucht et al. 2006; Wildner et al. 1998).
Two cell lines were created; Flp293A and 293 FLEX, both derived from 293 cells. The former pseudotyped with amphotropic and the latter with GaLV envelopes. Recently, a PG13-based murine producer cell line was also established using this strategy (Loew et al. 2009). A favorable chromosomal site for stable and high retroviral vector production is first identified and tagged. Due to the presence of two heterologous non-compatible FRT sites flanking the tagged retroviral genome, the subsequent re-use of this defined chromosomal site by means of RMCE is than performed to express a therapeutic gene. In order to select cell clones that underwent correct targeted integration reaction, the targeting viral vector contains a start codon that complements a transcriptionally inactive ATG-deficient selection marker after recombination.
The modular producer cell lines present several advantages: they are safer since integration of the vector within the packaging cell line was identified, the duration of the entire development process is much reduced as there is no need for screening and, in addition, production conditions are favorable due to the possibility of pre-adaptation of the master cell line to culture conditions and media. Thus, therapeutic virus production from bench to bedside becomes safer, faster, and cheaper (Coroadinha et al. 2010).
2.2. Lentiviral vectors
Similarly to retroviral vectors, the design of lentiviral vector packaging systems has evolved to minimize the risk of RCPs generation towards maximum safety. Currently, three generations of lentiviral vectors are considered. The first-generation (Naldini et al. 1996) closely resembles the three plasmid packaging system of simple retroviruses, except for the fact that the
In the second generation (Zufferey et al. 1997), the three plasmid system was maintained but all the accessory genes were deleted including
The development of a fourth generation of lentiviral vectors,
In addition to HIV-derived, other lentiviral vectors have been developed and reported to retain identical features to those of HIV’s based, including the ability to transduce non-dividing cells, high titers production, and the possibility to be pseudotyped with different envelope glycoproteins. These include lentiviral vectors based on SIV (simian immunodeficiency virus) (Pandya et al. 2001; Schnell et al. 2000), BIV (bovine immunodeficiency virus) (Matukonis et al. 2002; Molina et al. 2004), FIV (feline immunodeficiency virus) (Poeschla et al. 1998; Saenz and Poeschla 2004) and EAIV (equine infectious anaemia virus) (Balaggan et al. 2006; Mitrophanous et al. 1999; Stewart et al. 2009). Most of non-HIV derived lentiviral vectors have been reported to be
Contrarily to simple retroviral vectors, the cytotoxicity of some of the lentiviral proteins has hampered the establishment of stable cell lines constitutively expressing vector components. Therefore, the majority of the reported packaging cells for lentivirus manufacturing have been based on inducible systems that control the expression of the toxic proteins (for further details see section 3.1). Nevertheless, it is worth notice that transient production is still the main mean for lentiviral vector generation for both research and clinical purposes. Table 2 summarizes some of the available (stable) lentiviral vector packaging cell lines.
Except for the systems reported by and Ni et al. (2005), all the packaging cell lines for lentiviral vector production have been based on human 293 cells transformed with oncogenes such as the SV40 (simian vacuolating virus 40) large T antigen – 293T – or the Nuclear Antigen of Epstein-Barr Virus – 293EBNA.
For clinical application human 293 and 293T cells have been the exclusive cell substrates (Schweizer and Merten 2010). However, safety concerns arise from the fact that 90% of non-coding mobile sequences of the human genome are endogenous retrovirus and although most of them are defective, because of mutations accumulation, some are still active (Zwolinska 2006). Therefore, using human cell lines for the production of human retroviruses increases the chances of replicative-competent particles generation by homologous recombination (Pauwels et al. 2009). Also, the possibility of contamination with other human pathogens during the production process, poses additional hindrances to the use of human cells for biopharmaceuticals production, viral or not. In this context, the use of non-human cells would be strongly recommended, although the different glycosylation patterns of the envelope proteins could be an obstacle. For research purposes other human or monkey derived cells were tested (other 293 derived clones, HeLa, HT1080, TE671, COS-1, COS-7, CV-1), although most of them showed reduced vector production titers. Yet, COS-1 cells have shown to be capable of producing 3-4 times improved vector quality (expressed in infectious vector titer
|SODk||Human 293T||VSV-G||1.0 x 107||HIV-1 based||2nd||Tet-off||(Cockrell et al. 2006; Kafri et al. 1999; Xu et al. 2001)|
|293G||Human 293T||VSV-G||-||HIV-1 based||2nd||Tet-off||(Farson et al. 2001)|
|1.2 x 107|
1.6 x 106
8.5 x 106
|HIV-1 based||2nd||Continuous system.|
|(Ikeda et al. 2003)|
|NR||Human 293||VSV-G||3.5 x 107||HIV-1 based||2nd||Tet-off.|
Three level cascade gene regulation system: TRE → tat+rev → VSV-G+Gag-Pol. Codon-optimized
|(Ni et al. 2005)|
|REr1.35||Human 293T||VSV-G||1.8 x 105||HIV-1 based||3rd||Ecdysone inducible system. Codon-optimized ||(Pacchia et al. 2001)|
|293SF-pacLV||Human 293 EBNA||VSV-G||3.4 x 107||HIV-1 based||3rd||Tet-on||(Broussau et al. 2008)|
|PC48||Human 293T||VSV-G||7.4 x 105||EIAV based||3rd||Tet-on||(Stewart et al. 2009)|
|SgpG109||Human 293T||VSV-G||1 x 105||SIV-based||3rd||Ponasterone inducible system. Codon-optimized ||(Kuate et al. 2002)|
|GPRG||Human 293T||VSV-G||5 x 107||SIV-based||3rd||Introduction of vector by concatemeric array transfection. Tet-off||(Throm et al. 2009)|
3. Bioreaction platforms and production media
3.1. Stable vs. transient expression
Production platforms for lentiviral and retroviral vectors have been restrained to mammalian cells, typically murine or human derived, which are transfected with
Transient production, makes use of transfection methods to introduce the viral constructions, commonly cationic agents that complex with the negatively charged DNA, thus allowing it to be up-taken by the cell
Stable production relies on cell substrates in which the viral constructs where separately integrated into the cell genome, thus allowing their constitutive expression. Typically, the packaging functions are first inserted and after clonal selection of a high-level
Stable retroviral vector cell line development is a tedious and time consuming process which can take up to one year for a fully developed and characterized cell platform. However, it is compensated by obtaining continuously producing and highly consistent cell systems, prone to single-effort bioprocess and product characterization, a critical consideration for market approval.
Transient production is undoubtedly faster, when compared to the time frame necessary to develop a stable packaging cell line, presenting very competitive titers (up to 107 infectious vector
At a laboratory scale, transient production by plasmid transfection has been the first choice to cope with the cytotoxic proteins. For larger-scale production purposes, conditional packaging systems have been developed in which the expression of those is under the control of inducible promoters (Broussau et al. 2008; Farson et al. 2001; Kuate et al. 2002; Pacchia et al. 2001; Stewart et al. 2009). However transient transfection systems are, as discussed above, difficult to scale-up and do not fulfill adequate batch-to-batch variability standards; and, although the clinical trials currently using lentiviral vectors have been provided exclusively with transiently produced batches (Schweizer and Merten 2010), it is unlikely that a transient based systems will be approved when going from clinical to market. Conditional systems, on the other hand, require the addition/removal of the induction agents cumbering the production and requiring further down-stream stringency in processing of the viral preparations.
3.2. Stirred bioreaction vs. adherent cultures
It is widely accepted that stirred bioreaction systems using suspension cultures offer more advantages from the bioprocess view-point when compared to those under static/adherent conditions. The most evident advantage is the higher volumetric productivity, since suspension cultures in stirred systems present increased ratios of cell number
The first suspension system reported for high-titer retroviral vector production was based on a T-lymphoblastoid cell line using a third generation packaging construct, producing MLV derived retroviral vectors pseudotyped with amphotropic envelope: CEMFLYA cells (Pizzato et al. 2001). These cells were able to produce in the range of 107 infectious units
Despite the advances in the development of suspension cultures for stirred tank bioreactors and its clear advantage from the bioprocess view-point, retroviral and lentiviral vector manufacture for clinical batches has mainly been based on adherent static and preferably disposable systems, including large T-flasks, cell factories and roller bottles (Fig. 6) (Eckert et al. 2000; Merten et al. 2011; Przybylowski et al. 2006; Wikstrom et al. 2004). A good example is retroviral vector production at the National Gene Vector Laboratory, Indiana University, (Indianapolis, IN), a US National Institutes of Health initiative that has as main mission provide clinical grade vectors for gene therapy trials (Cornetta et al. 2005). Also for clinical-grade lentiviral vector production, the bioreaction system of choice has been Cell Factory or equivalent multitray systems (Merten et al. 2011; Schweizer and Merten 2010). These systems allow for 10 to 40 L vector production under GMP conditions, meeting the needs for initial trials, where usually a reduced number of patients are involved. In the future, if lentiviral and retroviral vector Gene Therapy products reach the market, it is still not clear if such systems will continue to be used. In fact, several restrictions arise from the use of disposable systems and bioreactors including the increase in the costs of solid waste disposal and consumables, in addition to low scalability and the single-use philosophy itself (Eibl et al. 2010). However, the low infectivity stability of retro and lentiviral vectors has hampered the perspective of the “thousand-liter” production systems’ for further storage. Nevertheless, significant efforts are being made to overcome this drawback including, at the bioprocess level, by developing storage formulations (Carmo et al. 2009a; Cruz et al. 2006) and at the viral vector design level, by developing mutant vectors with increased infectivity stability (Vu et al. 2008).
3.3. Bioreaction physicochemical parameters
The cell culture parameters used in the bioreaction may have a profound effect on the virus titer by affecting the cellular productivities, vector stability or both. Several studies have been performed analyzing the impact of physicochemical parameters such as pH, temperature, osmolarity, O2 and CO2 concentrations. The optimal cell culture parameters have been shown to be producer cell line and viral vector dependent.
The optimal pH range for retroviral vector production was found to be between 6.8 and 7.2 for FLY RD18 and Te FLY A7; outside this range the cell specific productivities were considerable lower (McTaggart and Al-Rubeai 2000; Merten 2004), while the retroviral vector was observed to be stable between pH of 5.5 and 8.0 in ecotropic pseudotyped vectors (Ye et al. 2003). Both retroviral vectors (MLV derived) and lentiviral vectors (HIV-1 derived), VSV-G pseudotyped, were stable at pH 7. The half-lives of both viral vectors at pH 6.0 and pH 8.0 markedly decrease to less than 10 minutes (Higashikawa and Chang 2001). The viral half-life is also dependent on the temperature: at lower temperatures the vector decay kinetics are lower (Le Doux et al. 1999). Therefore one strategy explored in the production of retroviral vectors has been the reduction of the culture temperatures (28-32º C). Some authors reported increases in vector production at lower temperature (Kaptein et al. 1997; Kotani et al. 1994; Le Doux et al. 1999; Lee et al. 1996). The reduction of the culture temperature from 37º C to 32º C extends vector stability allowing for the accumulation of more infectious virus and thus, increasing the volumetric titers. However, the increments are not always very significant as the temperature affects also the cell specific yields negatively. The improvement in the viral volumetric titer will be only observed if, the increase in the viral half-life is higher than the decrease in the cell specific production rate (Le Doux et al. 1999). Additionally, the viral vector inherent stability was also demonstrated to be lower when the viral vector was produced at 32º C instead of 37º C (Beer et al. 2003; Cruz et al. 2005). It was shown that the culture temperature affected the lipid viral membrane composition namely, the cholesterol content. The increase in cholesterol content was demonstrated to be inversely proportional to retroviral stability (Beer et al. 2003; Coroadinha et al. 2006c). Since enveloped virus, such as retrovirus and lentivirus, bud out of the host cells, they take part of the host cell lipidic membrane. Thus, the origin of the producer cell will have a pronounced effect on the viral particle stability and explain the discrepant results obtained for virus produced in different cells and at different temperatures. For PA317 cells, decreasing the production temperature from 37º C to 32º C resulted in an increase of 5-15 fold in the vector titers (Kaptein et al. 1997) while for PG13 lower titers were obtained (Reeves et al. 2000). The viral vector envelope glycoproteins also affect the viral particle inherent stability increasing the complexity and diversity of factors involved in the viral stability. Comparing lentiviral and retroviral vectors it was generally observed that HIV-1 derived vectors are more stable at 37º C and at higher temperatures than MLV derived vectors (Higashikawa and Chang 2001).
Augmenting the media osmolarity was also shown to be a valid strategy to increase retroviral vector titers in Te FLY A7 (Coroadinha et al. 2006c). This increment was correlated with higher cell specific productivities and higher inherent viral stability. The high osmotic pressure altered the cellular and viral envelope lipid membrane composition. High osmotic media were tested showing to induce a decrease in the cholesterol to phospholipids ratio in the viral membrane and thus conferring higher stability to the viral vectors produced (Coroadinha et al. 2006c). These results, together with the studies of production at lower culture temperatures, strengthen the importance of lipid metabolism in the production of enveloped virus.
CO2 gas concentration in the cultures did not affect virus production in packaging cell lines (Kotani et al. 1994; McTaggart and Al-Rubeai 2000). The dissolved oxygen levels used are between 20-80% and within this range do not affect viral production unless they became limiting to cell growth (Merten 2004).
3.4. Media composition and cell metabolic bottlenecks
Retroviral and lentiviral vector titers obtained in the production prior to purification are in the range of 106 to 107 infectious particles
The problems of low titers, short half-life and low ratios of infectious particles to total particles have been subject of intensive bioprocess research. However, the infection with wild type retroviruses, in particular HIV-1, is typically chronically and characterized by persistent but low titers of the infectious agents in the blood stream, with high amounts of non-infectious particles contaminants and with equivalently low half-lives (Perelson et al. 1996; Rusert et al. 2004). Therefore, retrovirus and lentiviral manufacture starts in disadvantage – when compared to other viral vectors – in what concerns to such parameters. Several strategies have been attempted to circumvent these “natural” drawbacks in packaging cell lines, including engineering mutant vectors with improved resistance features and understanding and optimizing the metabolic pathways leading to improved productivities. Studying the metabolic features driving to high titer performances has been one important work lines of research. Therefore, this section will mainly focus on the metabolic bottlenecks of viral vector production.
3.4.1. Serum supplemented vs. serum-free media
The supplementation of mammalian cell culture media with animal sera has been common practice in biomedical and biotechnological research, since it provides critical nutrients and factors that support cell growth and proliferation. However, the ill-defined composition and high batch-to-batch variability of serum together with its potential source of contaminations, hinders safety and standardization of cell cultures, making it a highly undesirable supplement in the production of biopharmaceuticals (Falkner et al. 2006). Also, in the case of retroviral and lentiviral vectors, serum needs to be removed from the medium and/or viral preparations to prevent immunological responses in the patients.
Retroviral and lentiviral vector manufacture has been reported to rely on considerable amounts (5-10% (v/v)) of animal sera in the culture medium; although some authors reported improved titers in short-term serum-free productions (Gerin et al. 1999a; McTaggart and Al-Rubeai 2000), the issue of serum dependence for retroviral and lentiviral vector production will be next discussed in the perspective of long-term cultures. The majority of the latest generation of packaging cell lines, specially the HEK293 (human embryonic kidney) derived ones, seem to require high concentrations of serum in the culture medium to support elevated viral productivities for long term culture (Chan et al. 2001; Gerin et al. 1999a; Gerin et al. 1999b; Pizzato et al. 2001; Rodrigues et al. 2009).
The need of serum for retroviral and lentiviral vector production has been mainly associated with the lipidic needs of packaging cell lines. Unless other supplements are added, serum is the only lipid source of the culture medium and, although cells should be able to sense lipid absence in the culture medium and activate biosynthetic pathways to stand up to lipid deprivation, the activation of lipid
The work done so far, addressing the issue of serum supplementation and infectious vector production, has mainly been focused on retroviral vectors, less attention has been paid to serum/lipid requirements in lentiviral vector production. Of notice is the work developed by B. Mitta et al (2005) in which optimal lentiviral production parameters were established, resulting in up to 132-fold improved productivities, and quality. The later is defined as the viral infectious titer (reflecting the number of transduction-competent lentiviral particles) relative to the number of total physical lentiviral particles produced (analysed by the levels of p24). A reduced-serum formulation was used and supplemented. Among others, lipid supplementation, included cholesterol, lecithin and chemically defined lipid concentrates. The lipid supplements were identified as the main responsibles for the improved viral productivities obtained.
In the case of lentiviral vectors, the short-term production periods associated with either the transient or conditional productions have not elucidated the extent of serum dependence in the production of high-infectious vector titers. Yet, the large majority of the current protocols for the production of lentiviral vectors still make use of 5 to 10% (v/v) of serum in the culture medium and up to now, only two publications have reported the production of lentiviral vectors under serum-free conditions (Ansorge et al. 2009; Broussau et al. 2008), both of them requiring lipid supplementation.
More recently, studies on the effects of adapting retroviral vector packaging cell lines to serum deprivation conditions and how it impacts infectious vector production have been performed. These studies identified differences in cell lipid metabolism as a requirement needed by the packaging cells to be able to adapt to serum deprivation: cells capable of activating
3.4.2. Sugar carbon source
Glucose has been the traditional sugar source employed in animal cell culture media and thus, the most used in the production of retroviral and lentiviral vectors. Together with glutamine, glucose is the major energy and carbon source in the culture medium. It is also the universal carbohydrate in animal cell culture, since glucose cellular transporters are present in the majority of the mammalian cell types. However, glucose is rapidly consumed and inefficiently metabolized to lactate which,
The use of alternative sugar sources to glucose is a possible strategy to decrease lactate production. Indeed, the use of fructose and galactose was shown to improve the retroviral production in Te FLY A7, Te FLY Ga18, PG13 and Tel CeB cell lines (Coroadinha et al. 2006a; Merten 2004). The lactate production decreased 2 to 6 fold in galactose and fructose media and the vector titers increased up to 8 fold. Both galactose and fructose consumption rates were lower than glucose in Te Fly A7, possible due to lower specificity of the sugar transporters expressed in these cells. The best results in terms of vector titers were obtained at high concentrations of fructose (15-25 g/L) (Coroadinha et al., 2006, Merten, 2004). Additionally to the metabolic shift induced by an alternative carbon source, an effect of high osmotic pressure can also be of relevance in the improvement of viral titers (see section 3.3). The increment of infectious titers observed at high sugar concentrations in Te Fly A7 was confirmed to be the result of higher cell specific productivities, higher vector stability and lower production of defective non-infective particles (Coroadinha et al., 2006a and 2006b) (Table 3).
molar ratio in viral particles
|Glucose 25 mM||335||0.18 ± 0.01||8 ± 0.7||0.53±0.03|
|Glucose 25 mM + sorbitol||450||0.80± 0.09||14± 1||0.33±0.01|
|Fructose 140 mM||450||1.0± 0.1||14±2||0.30±0.01|
Further metabolic studies were performed using 13C-NMR spectroscopy indicating changes in the lipid metabolism, namely higher synthesis of phospholipids (Coroadinha et al., 2006 and Amaral et al., 2008). These results show that packaging cell line metabolism deeply influences the productivity performances, in particular lipid biosynthesis, thus suggesting it to be an important target for further improve retroviral and possibly lentiviral vector titers.
No studies with alternative sugar sources have been reported with lentiviral vectors. Nevertheless, the above studies were performed with, Te671 and NIH 3T3 cells and most lentiviral vectors are produced in 293 derived cells.
4. Conclusions and outlook
Murine leukemia virus (MLV) derived vectors were the first viral vectors used in clinical trials and remain among the preferentially used vehicles for gene therapy applications due to their advantages relatively to other vectors. Lentiviral vectors have been developed more recently. From the therapeutic perspective they present the additional advantage of transducing non-dividing cells. From the manufacturing perspective lentiviral vectors present however, an additional difficulty as they contain cytotoxic proteins, requiring either the use of transient transfection or inducible systems. Both lentiviral and retroviral vectors are derived from virus belonging to the
The success of the application of retroviral vectors in phase I and II clinical trials is now moving the prospects to phase III trials. This will create momentum to increase the efforts in research related with retroviral vectors development and production due to the large amounts of vectors needed, and the stringent demands by the regulatory agencies. Lentiviral vectors in particular possess many of the characteristics of MLV retroviral vectors, and as mentioned present the additional advantages of being able to transduce quiescent cells. The diversity of human gene therapy as well as the possibility of patients being treated more than once with viral vectors, which are recognized by the adaptive immune system, leaves space to both alternative vector technologies. MLV present a large safety record in clinical application that cannot be discarded. Since MLV retroviral vectors are not derived from human viruses they also show reduced vector genome mobilization and recombination in the host-cell and pre-existing immune response against the retroviral vector particle. Additionally, they are simple to develop in terms of plasmid cloning, transfection and cell culture; and from the clinical perspective they can be easily produced at large scale from stable packaging cell lines with satisfactory yields. From the manufacturing point of view, HIV-1 derived vector still requires further optimization, particularly in what concerns cell line development. There is still less clinical experience with this vector and the results on the ongoing clinical trials will be certainly important for their improvement.
Thus the recent manufacturing strategies together with future innovations will certainly be important to increase productivity, stability, quality and safety of retroviral and lentiviral vectors for clinical applications.
The authors acknowledge the financial support received from the European Commission (CLINIGENE -LSHB-CT2006-018933) and the Fundação para a Ciência e a Tecnologia-Portugal (PTDC/EBB-BIO/100491/2008). Ana F. Rodrigues acknowledges FCT for her PhD grant (SFRH/BD/48393/2008).