Progress and Perspectives in the Development of Lentiviral Vector Producer Cells

After two decades of clinical trials, gene therapy demonstrated effectiveness in the treatment of a series of diseases. Currently, several gene therapy products are approved and used in the clinic. Lentiviral vectors (LVs) are one of the most used transfer vehicles to deliver genetic material and the vector of choice to modify hematopoietic cells to correct primary immunodeficiencies, hemoglobinopathies, and leukodystrophies. LVs are also widely used to modify T cells to treat cancers in immunotherapies (e.g., chimeric antigen receptors T cell therapies, CAR-T). In genome editing, LVs are used to deliver sequence-specific designer nucleases and DNA templates. The approval LV gene therapy products (e.g., Kymriah, for B-cell Acute lymphoblastic leukemia treatment; LentiGlobin, for β-thalassemia treatment) reinforced the need to improve their bioprocess manufacturing. The production has been mostly dependent on transient transfection. Production from stable cell lines facilitate GMP compliant processes, providing an easier scale-up, reproducibility and cost-effectiveness. The establishment of stable LV producer cell lines presents, however, several difficulties, with the cytotoxicity of some of the vector proteins being a major challenge. Genome editing technologies pose additional challenges to LV producer cells. Herein the major bottlenecks, recent achievements, and perspectives in the development of LV stable cell lines are revised.     

Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses

BACKGROUND: The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. METHODS: Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. RESULTS: The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. CONCLUSIONS: The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.     

Identification of a replication-defective herpes simplex virus for recombinant adeno-associated virus type 2 (rAAV2) particle assembly using stable producer cell lines

BACKGROUND: The development of stable producer cell lines for recombinant adeno-associated virus (rAAV) assembly is a strategy followed by many groups to develop scalable production methods suitable for good manufacturing practice (GMP) requirements. The major drawback of this method lies in the requirement for replicating adenovirus (Ad) for rAAV assembly. In the present study, we analyzed the ability of several replication-defective herpes simplex type 1 (HSV-1) helper viruses to induce rAAV2 particle production from stable producer cell lines. METHODS: Several stable rAAV producer cell clones were infected with wild-type and replication-defective HSV strains and analyzed for rep-cap gene amplification, viral protein synthesis and rAAV titers achieved. In vivo analysis following rAAV injection in the murine brain was also conducted to evaluate the toxicity and biopotency of the rAAV stocks. RESULTS: We demonstrated that an HSV strain mutated in the UL30 polymerase gene could efficiently be used in this context, resulting in rAAV titers similar to those measured with wild-type HSV or Ad. Importantly, with respect to clinical developments, the use of this mutant resulted in rAAV stocks which were consistently devoid of contaminating HSV particles and fully active in vivo in the murine central nervous system with no detectable toxicity. CONCLUSIONS: This study, together with our previous report describing a rAAV chromatography-based purification process, contributes to the definition of an entirely scalable process for the generation of rAAV particles.     

Packaging Cells Based on Inducible Gene Amplification for the Production of Adeno-Associated Virus Vectors

Although vectors based on adeno-associated virus (AAV) offer several unique advantages, their usage has been hampered by the difficulties encountered in vector production. In this report, we describe a new AAV packaging system based on inducible amplification of integrated helper and vector constructs containing the simian virus 40 (SV40) replication origin. The packaging and producer cell lines developed express SV40 T antigen under the control of the reverse tetracycline transactivator system, which allows inducible amplification of chromosomal loci linked to the SV40 origin. Culturing these cells in the presence of doxycycline followed by adenovirus infection resulted in helper and vector gene amplification as well as higher vector titers. Clonal producer cell lines generated vector titers that were 10 times higher than those obtained by standard methods, with approximately 10⁴ vector particles produced per cell. These stocks were free of detectable replication-competent virus. The lack of a transfection step combined with the reproducibility of stable producer lines makes this packaging method ideally suited for the large-scale production of vector stocks for human gene therapy.     

Development of a perfusion process for continuous lentivirus production using stable suspension producer cell lines

The large-scale production of clinical-grade lentiviral vectors (LVs) for gene therapy applications is a remaining challenge. The use of adherent cell lines and methods like transient transfection are cost-intensive and hamper process scalability as well as reproducibility. This study describes the use of two suspension-adapted stable packaging cell lines, called GPRGs and GPRTGs, for the development of a scalable and serum-free LV production process. Both stable packaging cell lines are based on an inducible Tet-off system, thus requiring doxycycline removal for initiation of the virus production. Therefore, we compared different methods for doxycycline removal and inoculated three independent 5 L bioreactors using a scalable induction method by dilution, an acoustic cell washer and manual centrifugation. The bioreactors were inoculated with a stable producer cell line encoding for a LV carrying a clinically relevant gene. LV production was performed in perfusion mode using a cell retention device based on acoustic wave separation. Comparable cell-specific productivities were obtained with all three methods and cumulative functional yields up to 6.36 × 10¹¹ transducing units per bioreactor were generated in a 234-h long process, demonstrating the usability of stable Tet-off cell lines for an easily scalable suspension process. Remarkably, cell viabilities >90% were maintained at high cell densities without compromising productivity throughout the whole process, allowing to further extend the process time. Given its low effects of toxicity during virus production, the presented cell lines are excellent candidates to develop a fully continuous LV production process to overcome the existing bottlenecks in LV manufacturing.     

Generation of stable retrovirus packaging cell lines after transduction with herpes simplex virus hybrid amplicon vectors

Background

A number of properties have relegated the use of Moloney murine leukemia virus (Mo‐MLV)‐based retrovirus vectors primarily to ex vivo protocols. Direct implantation of retrovirus producer cells can bypass some of the limitations, and in situ vector production may result in a large number of gene transfer events. However, the fibroblast nature of most retrovirus packaging cells does not provide for an effective distribution of vector producing foci in vivo, especially in the brain. Effective development of new retrovirus producer cells with enhanced biologic properties may require the testing of a large number of different cell types, and a quick and efficient method to generate them is needed.

Methods

Moloney murine leukemia virus (Mo‐MLV) gag‐pol and env genes and retrovirus vector sequences carrying lacZ were cloned into different minimal HSV/AAV hybrid amplicons. Helper virus‐free amplicon vectors were used to co‐infect glioma cells in culture. Titers and stability of retrovirus vector production were assessed.

Results

Simultaneous infection of two glioma lines, Gli‐36 (human) and J3T (dog), with both types of amplicon vectors, generated stable packaging populations that produced retrovirus titers of 0.5–1.2×10⁵ and 3.1–7.1×10³ tu/ml, respectively. Alternatively, when cells were first infected with retrovirus vectors followed by infection with HyRMOVAmpho amplicon vector, stable retrovirus packaging populations were obtained from Gli‐36 and J3T cells producing retrovirus titers comparable to those obtained with a traditional retrovirus packaging cell line, ΨCRIPlacZ.

Conclusions

This amplicon vector system should facilitate generation of new types of retrovirus producer cells. Conversion of cells with migratory or tumor/tissue homing properties could result in expansion of the spatial distribution or targeting capacity, respectively, of gene delivery by retrovirus vectors in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus-based lentiviral vectors

Background

Lentiviral vectors allow gene transfer into non‐dividing cells. Further development of these vector systems requires stable packaging cell lines that enable adequate safety testing.

Methods

To generate a packaging cell line for vectors based on simian immunodeficiency virus (SIV), expression plasmids were constructed that contain the codon‐optimized gag‐pol gene of SIV and the gene for the G protein of vesicular stomatitis virus (VSV‐G) under the control of an ponasterone‐inducible promoter. Stable cell lines expressing these packaging constructs were established and characterized.

Results

The RT activity and vector titers of cell clones stably transfected with the inducible gag‐pol expession plasmid could be induced by ponasterone by more than a factor of 1000. One of these clones was subsequently transfected with the ponasterone‐inducible VSV‐G expression plasmid to generate packaging cells. Clones of the packaging cells were screened for vector production by infection with an SIV vector and subsequent induction by ponasterone. In the supernatant of selected ponasterone‐induced producer clones vector titers of more than 1×10⁵ transducing units/ml were obtained. Producer cell clones were stable for at least five months, as tested by vector production.

Conclusions

The packaging cells described should be suitable for most preclinical applications of SIV‐based vectors. By avoiding regions of high homology between the vector and the packaging constructs, the design of the SIV packaging cell line should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the SIV vector constructs that can be packaged. Copyright © 2002 John Wiley & Sons, Ltd.

     

Packaging system for rapid production of murine leukemia virus vectors with variable tropism.

A method for rapidly producing helper-free murine leukemia virus (MLV) without using packaging cell lines is described. Viruses bearing ecotropic or amphotropic MLV or Rous sarcoma virus envelope glycoprotein and containing various retroviral vector genomes have been prepared with titers 30 to 40-fold higher than those produced by transient transfection of standard packaging cells. This system can be used to alter the cellular tropism of MLV by incorporating other envelope glycoproteins and to prepare retroviral vector stocks without establishing stable producer cell lines. This method will be particularly useful for preparing viruses that encode toxic proteins and for the rapid analysis of panels of mutant envelope glycoproteins.     

Human immunodeficiency virus type 1 vectors with alphavirus envelope glycoproteins produced from stable packaging cells

Alphavirus glycoproteins have broad host ranges. Human immunodeficiency virus type 1 (HIV-1) vectors pseudotyped with their glycoproteins could extend the range of tissues that can be transduced in both humans and animal models. Here, we established stable producer cell lines for HIV vectors pseudotyped with alphavirus Ross River virus (RRV) and Semliki Forest virus (SFV) glycoproteins E2E1. RRV E2E1-stable clones could routinely produce high-titer pseudotyped vectors for at least 5 months. SFV E2E1-stable clones, however, produced relatively low titers. We examined the properties of RRV E2E1-pseudotyped vectors [HIV-1(RRV)] and compared them with amphotropic murine leukemia virus Env- and vesicular stomatitis virus glycoprotein G-pseudotyped vectors. HIV-1(RRV) displayed a number of characteristics which would be advantageous in ex vivo and in vivo experiments, including resistance to inactivation by heat-labile components in fresh human sera and thermostability at 37 degrees C. Upon single-step concentration by ultracentrifugation of HIV-1(RRV), we could achieve vector stocks with titers up to 6 x 10(7) IU/ml. HIV-1(RRV) efficiently transduced cells from several different species, including murine primary dendritic cells, but failed to transduce human and murine T cells as well as human hematopoietic stem cells (HSC). These results indicate that HIV-1(RRV) could be used in a number of applications including animal model experiments and suggest that expression of RRV cellular receptors is limited or absent in certain cell types such as T cells and human HSC.     

Production of lentiviral vectors with enhanced efficiency to target dendritic cells by attenuating mannosidase activity of mammalian cells

Background  

Dendritic cells (DCs) are antigen-presenting immune cells that interact with T cells and have been widely studied for vaccine applications. To achieve this, DCs can be manipulated by lentiviral vectors (LVs) to express antigens to stimulate the desired antigen-specific T cell response, which gives this approach great potential to fight diseases such as cancers, HIV, and autoimmune diseases. Previously we showed that LVs enveloped with an engineered Sindbis virus glycoprotein (SVGmu) could target DCs through a specific interaction with DC-SIGN, a surface molecule predominantly expressed by DCs. We hypothesized that SVGmu interacts with DC-SIGN in a mannose-dependent manner, and that an increase in high-mannose structures on the glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is known that 1-deoxymannojirimycin (DMJ) can inhibit mannosidase, which is an enzyme that removes high-mannose structures during the glycosylation process. Thus, we investigated the possibility of generating LVs with enhanced capability to modify DCs by supplying DMJ during vector production.     

Improvement of avian leukosis virus (ALV)-based retrovirus vectors by using different cis-acting sequences from ALVs.

Production and expression of double-expression vectors which transduce both Neo(r) and lacZ genes and are based on the structure of avian leukosis virus were enhanced by using cis-acting sequences (long terminal repeats and noncoding sequences) from Rous-associated virus-1 and Rous-associated virus-2 rather than those of avian erythroblastosis virus previously used in our constructs. Polyclonal producer cells obtained after transfection of these vectors into the Isolde packaging cell line gave rise to titers as high as 3 x 10(5) lacZ CFU/ml, whereas it was possible to isolate clones of producer cells giving rise to titers of more than 10(6) resistance focus-forming units per ml.     

Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses

We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency.     

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.     

Optimization of environmental factors for the production and handling of recombinant retrovirus

Certain steps from the production to infection of the amphotrophic retroviral vector, MFG-LacZ, were optimized and the factors that affect retroviral titers were analyzed. Retroviral vector titers were highest when the culture supernatant was harvested 3 days after the producer cells had reached confluence. About a 2-fold increase in vector production was achieved at 32°C compared to that at 37°C. Low serum concentrations had no significant effect on the titers of virus produced by the CRIP cell line. Retroviral vectors were stable at 4°C but very unstable at 37°C and were quite sensitive to freezing and thawing. About 30%–50% of viral infectivity was lost during the thawing step and the loss was not recovered by the addition of commonly used cryoprotectants. Increase in viral exposure time for infection to target NIH3T3 cells was linearly proportional to the retroviral titer for up to 15 h. In addition, using DEAE-dextran in place of polybrene as a polycation during infection enhanced infection efficiency about 3-fold. The retrovirus was robust to simple ultrafiltration and its titer could be easily concentrated 16-fold. Taken together, our data suggest that at least a 100-fold increase in titer can be achieved with simple optimization.     

The effect of viral regulatory protein expression on gene delivery by human immunodeficiency virus type 1 vectors produced in stable packaging cell lines.

We describe the generation of stable human immunodeficiency virus type 1 (HIV-1)-packaging lines that constitutively express high levels of HIV-1 structural proteins in either a Rev-dependent or a Rev-independent fashion. These cell lines were used to assess gene transfer by using an HIV-1 vector expressing the hygromycin B resistance gene and to study the effects of Rev, Tat, and Nef on the vector titer. The Rev-independent cell lines were created by using gag-pol and env expression vectors that contain the Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). Vector titers approaching 10(4) CFU/ml were routinely obtained with these cell lines, as well as with the Rev-dependent cell lines, with HeLa-CD4 cells as targets. The presence of Nef and Tat in the producer cell each increased the vector titer 5- to 10-fold. Rev, on the other hand, was absolutely essential for gene transfer, unless the MPMV CTE was present in the vector. In that case, by using the Rev-independent cell lines for packaging, Rev could be completely eliminated from the system without a reduction in vector titer.     

Lentiviruses inefficiently incorporate human parainfluenza type 3 envelope proteins

We have previously shown that the envelope glycoproteins of human parainfluenza type 3 (HPIV3), F and HN, are able to pseudotype lentiviruses, but the titers of these viruses are too low for use in clinical gene transfer. In this study we investigated the cause of these low titers. We compared the mRNA and protein expression levels of HN and F in transfected cells and in cells infected with wild-type HPIV3. Transfected cells contained similar levels of HN and F cytosolic mRNA, but fewer cell-surface HN and F proteins (3.8- and 1.3-fold less, respectively), than cells infected with wild-type HPIV3. To increase expression of HN in transfected cells, we codon-optimized HN and used it to transfect lentivirus producer cells. Cell surface expression of HN, as well as the amount of HN incorporated into virus particles, increased two- to threefold. Virus titers increased 1.2- to 6.4-fold, and the transduction efficiency of polarized MDCK cells via their apical surfaces increased 1.4-fold. Interestingly, even though codon optimization improved the expression levels of HN and virus titers, we found that HPIV3 pseudotyped viruses contained about 14-fold fewer envelope proteins than lentiviruses pseudotyped with the amphotropic envelope protein. Taken together, our findings suggest that titers are low, not because virus producer cells express levels of HPIV3 envelope proteins that are too low, but because too few of these proteins are incorporated by the lentiviruses for them to be able to efficiently transduce cells.