Lentiviral Production Research Articles

Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes.

Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B, and SPINK6) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with LDAH knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of GBP3, BPIFC, and LDAH increased LV titer by ∼8.33-fold, and knockout (or knockdown) of GBP3, NHLRC1, and NHLRC3 increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to4.64×107TU mL-1in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38×107TU mL-1in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of>1×107TUmL-1day-1for up to 29 consecutive days in a non-optimized 5L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Evaluation of Lentiviral Based Gene Delivery System in Adherent and Suspension in vitro Cell Models

Lentiviruses are a highly robust gene delivery system capable of in vitro and in vivo gene transfer into multiple cell types. Recent fourth-generation lentiviral systems have been designed for enhanced safety, however, the increased recombination events required to produce infective lentiviral particles may reduce production efficiency. A set protocol for all types of target cells is not recommended and optimization of conditions for gene transfer into different target cells is required. In this study, we aim to evaluate the efficiency and reproducibility of lentiviral production using a fourth-generation lentiviral packaging system and identify optimal parameters for successful transduction in two different cell models, adherent and suspension cells. Lentiviral production, effect of viral volume, sustained gene expression and transduction adjuvants on adherent and suspension gene- cell models were evaluated. Transfection and transduction efficiency of lentiviruses was evaluated by fluorescence microscopy and flow cytometry. This study demonstrates that production of green fluorescent protein (GFP)- lentiviruses using the fourth-generation lentiviral packaging is consistent and reproducible. Optimal transduction of adherent cell types is achieved at lower multiplicity of infection (MOI) compared to suspension cells and produces GFP-expressing cells with higher intensity. Expression of GFP is sustained in all cell types over multiple passages. Polycation DEAE-dextran was determined to improve transduction in suspension cells, however, provides similar transduction efficiency as polybrene in adherent cells. In conclusion, fourth generation lentiviral system reproducibly generates high titre lentiviruses capable of infecting multiple cell types, however transduction protocols for different cell types require further optimization.

Use of Lentivirus-Based Method for Establishing TK6 Human Cell Lines Expressing Cytochrome P450 and its Applications in Genotoxicity Testing.

The human lymphoblastoid cell line TK6 stands out as the most widely employed human cell line in genotoxicity testing, as recommended by various testing guidelines for in vitro assessments. Nevertheless, like many testing cell lines, TK6 lacks functional phase I drug-metabolizing enzymes crucial for chemical genotoxicity evaluations. This protocol introduces a lentivirus-based methodology for establishing a panel of TK6-derived cell lines, each expressing one of 14 cytochrome P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7). The utilization of a lentiviral expression system ensures stable transduction, offering notable advantages such as sustained transgene expression, high transduction efficiency, positive selection feasibility, and user-friendly application. Additionally, we present a detailed procedure for validating the enhanced expression of each CYP in the established cell lines through real-time PCR, western blotting, and mass spectrometry analysis. Lastly, we exemplify the application of these CYP-expressing TK6 cell lines in genotoxicity testing, employing a flow-cytometry-based in vitro micronucleus test. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Lentivirus production and transduction for TK6 cells Support Protocol: Selecting a single clone of CYP-expressing TK6 cells Basic Protocol 2: Validation of CYP expression in TK6 cell lines Basic Protocol 3: Application of transduced cell lines in flow-cytometry-based micronucleus assay.

A Pseudovirus-Based Neutralization Assay for SARS-CoV-2 Variants: A Rapid, Cost-Effective, BSL-2-Based High-Throughput Assay Useful for Vaccine Immunogenicity Evaluation.

Neutralizing antibody responses from COVID-19 vaccines are pivotal in conferring protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Effective COVID-19 vaccines and assays measuring neutralizing antibodies against emerging variants (i.e., XBB.1.5, XBB.1.16, and XBB.2.3) are needed. The use of biosafety level (BSL)-3 laboratories for live virus assays results in higher costs and a longer turnaround time; therefore, a BSL-2-based pseudovirus neutralization assay (PNT) was developed. The pseudoviruses were produced by cotransfecting cells with plasmids encoding a lentiviral backbone-expressing luciferase reporter; non-surface proteins for lentiviral production; and ancestral or Omicron (BA.1 and BA.5) SARS-CoV-2 spike (S) proteins. The PNT was developed and optimized in dose and kinetics experiments. The representative serum samples (COVID-19-convalescent or NVX-CoV2373-vaccinated participants enrolled in the 2019nCoV-101 trial) demonstrated a wide dynamic range. The neutralization data showed robust correlation with validated anti-recombinant spike IgG levels and angiotensin-converting enzyme 2 inhibition titers (ancestral). This assay is suitable for measurement of the neutralization ability in clinical samples from individuals infected with SARS-CoV-2 or immunized with a COVID-19 vaccine. The results suggest that this PNT provides a lower cost, high-throughput, rapid turnaround alternative to BSL-3-based microneutralization assays and enables the discovery and development of effective vaccines against emerging variants.

Lentiviral Production Platform.

Lentiviral-mediated transfection technique is a powerful tool for gene modification in preclinical studies. By using this technique, the desired gene modification can be achieved easily in immune cells, nondividing, and terminally differentiated cells, including hematopoietic stem cells, neurons, and even tumor cells, which other viral vectors cannot do. The main considerations of therapeutic gene delivery using a lentiviral system are the risk of insertional mutagenesis and the immune reaction elicited by infected cells. Although some biosafety concerns need to be addressed before clinical trials in rheumatoid arthritis, the lentiviral system targeting therapeutic targets has been widely used for in vivo gene transfer in animal models. In this chapter, the protocols for production of viral particles and viral concentration are provided. As an alternative utilization, this lentiviral production platform could also be employed to produce a pseudotype severe acute respiratory syndrome-related coronavirus 2 in which the spike glycoprotein of SARS-CoV-2 was incorporated into pseudovirions for viral study.

An optimized & streamlined approach for upstream & downstream lentiviral production

An optimized & streamlined approach for upstream & downstream lentiviral production

FAM46C Is an Interferon-Stimulated Gene That Inhibits Lentiviral Particle Production by Modulating Autophagy.

FAM46C is a multiple myeloma (MM) tumor suppressor whose function is only starting to be elucidated. We recently showed that in MM cells FAM46C triggers apoptosis by inhibiting autophagy and altering intracellular trafficking and protein secretion. To date, both a physiological characterization of FAM46C role and an assessment of FAM46C-induced phenotypes outside of MM are lacking. Preliminary reports suggested an involvement of FAM46C with regulation of viral replication, but this was never confirmed. Here, we show that FAM46C is an interferon-stimulated gene and that the expression of wild-type FAM46C in HEK-293T cells, but not of its most frequently found mutant variants, inhibits the production of both HIV-1-derived and HIV-1 lentiviruses. We demonstrate that this effect does not require transcriptional regulation and does not depend on inhibition of either global or virus-specific translation but rather mostly relies on FAM46C-induced deregulation of autophagy, a pathway that we show to be required for efficient lentiviral particle production. These studies not only provide new insights on the physiological role of the FAM46C protein but also could help in implementing more efficient antiviral strategies on one side and lentiviral particle production approaches on the other. IMPORTANCE FAM46C role has been thoroughly investigated in MM, but studies characterizing its role outside of the tumoral environment are still lacking. Despite the success of antiretroviral therapy in suppressing HIV load to undetectable levels, there is currently no HIV cure, and treatment is lifelong. Indeed, HIV continues to be a major global public health issue. Here, we show that FAM46C expression in HEK-293T cells inhibits the production of both HIV and HIV-derived lentiviruses. We also demonstrate that such inhibitory effect relies, at least in part, on the well-established regulatory role that FAM46C exerts on autophagy. Deciphering the molecular mechanism underlying this regulation will not only facilitate the understanding of FAM46C physiological role but also give new insights on the interplay between HIV and the cellular environment.

Serum-free lentiviral vector production is compatible with medium-resident nuclease activity arising from adherent HEK293T host cells engineered with a nuclease-encoding transgene

At present lentiviral vector production for cell and gene therapy commonly involves transient plasmid transfection of mammalian cells cultivated in serum-containing media and addition of exogenous nuclease to reduce host cell and plasmid DNA impurities. Switching from serum-containing media to chemically-defined, serum free media, and minimising the number of process additions, are both increasingly regarded as necessary steps for simplifying and potentially automating lentiviral vector bioprocessing in future. Here we adapted human embryonic kidney 293T (HEK293T) cells to grow in serum-free media and also modified these cells with transgenes designed to encode a secreted nuclease activity. Stable transfection of HEK293T cells with transgenes encoding the Staphylococcus aureus nuclease B (NucB) open reading frame with either its native secretion signal peptide, the murine Igκ chain leader sequence or a novel viral transport fusion protein, all resulted in qualitatively detectable nuclease activity in serum-free media. Serum-free transient transfection of human embryonic kidney HEK293T cells stably harbouring the transgene for NucB with its native secretion signal produced active lentivirus in the presence of medium-resident nuclease activity. This lentivirus material was able to transduce the AGF-T immortal T cell line with a green fluorescent protein reporter payload at a level of 2.05 × 105 TU/mL (±3.34 × 104 TU/mL). Sufficient nuclease activity was present in 10 μL of this unconcentrated lentivirus material to degrade 1.5 μg DNA within 2 h at 37 °C, without agitation - conditions compatible with lentivirus production. These observations demonstrate that lentiviral vector production, by transient transfection, is compatible with host cells harbouring a nuclease transgene and evidencing nuclease activity in their surrounding growth media. This work provides a solid basis for future investigations, beyond the scope of this present study, in which commercial and academic groups can apply this approach to therapeutic payloads and potentially omit exogenous nuclease bioprocess additions.

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.

CRISPR/Cas9-Mediated Gene Knockout in Cells and Tissues Using Lentivirus.

CRISPR-Cas9 has become a powerful and popular gene editing tool. However, successful application of this tool in the lab can still be quite daunting to many newcomers to molecular biology, mostly because it is a relatively lengthy process involving multiple steps with variations of each step. Here, we provide a reliable, stepwise, and newcomer-friendly protocol to knock out a target gene in wild-type human fibroblasts. This protocol involves sgRNA design using CRISPOR, construction of an "all-in-one" vector expressing both sgRNA and Cas9 using Golden Gate cloning, streamlined production of high-titer lentiviruses in 1 week after molecular cloning, and transduction of cells to generate a knockout cell pool. We further introduce a protocol for lentiviral transduction of ex vivo mouse embryonic salivary epithelial explants. In summary, our protocol is useful for new researchers to apply CRISPR-Cas9 to generate stable gene knockout cells and tissue explants using lentivirus. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning sgRNA in plasmid vector containing Cas9 encoding sequence using golden gate cloning Basic Protocol 3: Lentivirus packaging Basic Protocol 4: Lentivirus transduction of cells Basic Protocol 5: Lentivirus transduction of salivary gland epithelial buds.

Directed Differentiation of Human iPSCs into Microglia-Like Cells Using Defined Transcription Factors.

The generation of a homogeneous population of microglia from human induced pluripotent stem cells (hiPSCs) is crucial to modeling neurological disorders, as well as the carrying out of drug screening and toxicity testing. Here, we provide a stepwise protocol for the simple, robust, and efficient differentiation of hiPSCs into microglia-like cells (iMGs) by overexpression of SPI1 and CEBPA. This protocol details hiPSC culture, lentivirus production, lentivirus delivery, and, finally, the differentiation and validation of the iMG cells.

Small RNA interfering effects in chicken embryonic fibroblasts using two sites 8664/p201 and 2122/pll3.7 of chicken s1p1/edg 1 gene

Introduction: Currently, short interfering RNA (siRNA) is an experimental tool in molecular biology to study gene function in cell culture and in vivo in model of organisms (mouse and chicken embryos, etc.) Method: Lentivirus production technique by human embryonic kidney 293T cell lines (HEK293T) with two (2) sites 8664/P201 and 2122/Pll.3.7 of the sphingosine 1 phosphate receptor gene and endothelial differentiation 1 gene (S1P1/ Chicken EDG1) was used to show the effects of small interfering RNA in chicken embryonic fibroblasts during embryogenesis. After preparation of plasmids 8664/P201 and 2122/Pll3.7, lentivirus packaging, transfection with 2xHBS, virus titration and chicken egg preparation, microinjection of lentivirus was performed in the embryos chicken. Results: The results obtained after the microinjection of lentivirus into chicken embryos from the second day to the fourth day of incubation showed the effects of small interfering RNA in the embryonic development of chicken materialized by bleeding in the yolk sac of embryos therefore a decrease in gene function. Conclusion: Previous studies have already proven that expression of small interfering RNA decreases in embryos of certain vertebrates such as mammals.

Genome-wide profiling of retroviral DNA integration and its effect on clinical pre-infusion CAR T-cell products

BackgroundClinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and γ-retroviral vectors are the most commonly used vectors in the manufacturing process. However, the integration pattern of these viral vectors and subsequent effect on CAR T-cell products is still unclear.MethodsWe used a modified viral integration sites analysis (VISA) pipeline to evaluate viral integration events around the whole genome in pre-infusion CAR T-cell products. We compared the differences of integration pattern between lentiviral and γ-retroviral products. We also explored whether the integration sites correlated with clinical outcomes.ResultsWe found that γ-retroviral vectors were more likely to insert than lentiviral vectors into promoter, untranslated, and exon regions, while lentiviral vector integration sites were more likely to occur in intron and intergenic regions. Some integration events affected gene expression at the transcriptional and post-transcriptional level. Moreover, γ-retroviral vectors showed a stronger impact on the host transcriptome. Analysis of individuals with different clinical outcomes revealed genes with differential enrichment of integration events. These genes may affect biological functions by interrupting amino acid sequences and generating abnormal proteins, instead of by affecting mRNA expression. These results suggest that vector integration is associated with CAR T-cell efficacy and clinical responses.ConclusionWe found differences in integration patterns, insertion hotspots and effects on gene expression vary between lentiviral and γ-retroviral vectors used in CAR T-cell products and established a foundation upon which we can conduct further analyses.

A feasibility study of different commercially available serum-free mediums to enhance lentivirus and adeno-associated virus production in HEK 293 suspension cells.

The online version contains supplementary material available at 10.1007/s10616-022-00551-1.

XIII EHOC 2022 / CELLULAR THERAPY: CAR T-CELLS IN HEMATOLOGICAL MALIGNANCIES

ARI-0001 [systematically named Varnimcabtagene autoleucel (var-cel), a second generation anti-CD19 chimeric antigen receptor (CAR) T-cell] granted local use authorization (under the rule of “hospital exemption”, HE) by the AEMPS (Spanish drug agency = Agencia Española de Medicinamentos y Productos Sanitarios) and just a little more than half-year ago (December 2021) PRIME (Priority Medicine) designation by the EMA (European Mediciness Agency) for patients >25 years old with relapsed or refractory (R/R) B cell acute lymphoblastic leukaemia (B-ALL). The authorization is based on the results of a phase 1 clinical trial (NCT03144583), but additional patients (already reimbursed by Spanish Health System), new clinical trials or compassionate uses with ARI-0001, have been produced and infused in our Hospital Clínic de Barcelona or our pediatric partner, Hospital Sant Joan de Déu. Although HE for adult ALL patients and compassionate uses (next to indicated commercial products that authorized our center) allow us to use CART19 therapy for treating our patients (our real aim of this development), the good clinical results, and petitions of different centers all around the world (specially from places where commercial products are not available) encouraged us to consider how we should proceed to extend our product to other patients.Our Academic proposal is the result of the work of a multidisciplinary team, a point-of-care (PoC) procedure based on a well stablish protocol in a commercially available bioreactor and our home-developed lentivirus.All the elements of our proposal follow the GMP standards, strictly controlled by the AEMPS and the regulations for Advanced Therapy Medicinal Products (ATMPs) of the EMA; although the product could be developed in our clean-rooms at Barcelona, our aim is to share procedures to allow production as a real PoC product, looking for partners that can reproduce all steps next to the patient. This multi-site cell production has been already accomplished with success in several clinical trials, while for a homogeneous lentiviral production, we decided (by now) to use facilities centralized in our university hospital. We expect to obtain first local authorization for this multicenter production in Spain, and later by EMA and other regulators (India). In fact, this experience is also supported by developing a clinical trial with 60 multiple myeloma patients under the treatment of a new own CART-BCMA (ARI-0002h).We are convinced that it is a possible model, although most of the huge number of rules are mainly thought for pharma-companies and are not easily implemented by Academic entities. But if we want to have the best treatments for our patients, to find solutions with real options for Academic ATMPs developments is the only way to arrive where the commercial companies, the health systems and in general countries will not be able to arrive for different reasons (difficult recover of investments by complex reimbursement, low level of patients, no-sustainable expenses and procedures for economic and ecologic reason, ...).

Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions.

There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting accumulation of full-length viral genomes, subgenomic RNAs and structural proteins. In ectopic expression models, we observed that 6-TG increased the electrophoretic mobility of Spike from diverse betacoronaviruses, matching the effects of enzymatic removal of N-linked oligosaccharides from Spike in vitro. SARS-CoV-2 virus-like particles (VLPs) harvested from 6-TG-treated cells were deficient in Spike. 6-TG treatment had a similar effect on production of lentiviruses pseudotyped with SARS-CoV-2 Spike, yielding pseudoviruses deficient in Spike and unable to infect ACE2-expressing cells. Together, these findings from complementary ectopic expression and infection models strongly indicate that defective Spike trafficking and processing is an outcome of 6-TG treatment. Using biochemical and genetic approaches we demonstrated that 6-TG is a pro-drug that must be converted to the nucleotide form by hypoxanthine phosphoribosyltransferase 1 (HPRT1) to achieve antiviral activity. This nucleotide form has been shown to inhibit small GTPases Rac1, RhoA, and CDC42; however, we observed that selective chemical inhibitors of these GTPases had no effect on Spike processing or accumulation. By contrast, the broad GTPase agonist ML099 countered the effects of 6-TG, suggesting that the antiviral activity of 6-TG requires the targeting of an unknown GTPase. Overall, these findings suggest that small GTPases are promising targets for host-targeted antivirals.

Development of a lentivirus-mediated gene therapy targeting HIV-1 RNA to eliminate HIV-1-infected cells

Only two patients have ever been cured of HIV-1. The latent HIV-1 reservoir is the major roadblock to this and necessitates lifelong therapy. In the ‘shock and kill’ approach to eliminate cells harbouring dormant virus the latency reversing agent (LRA) vorinostat increased HIV-1 RNA levels, but this failed to enable a reduction in reservoir size by immune- or virus-mediated cytotoxicity. To leverage LRAs’ ability to heighten HIV-1 transcription, we are developing a ‘kill’ strategy that hijacks the HIV-1 splicing process with a therapeutic trans-splicing RNA, encoding an incomplete Herpes simplex virus thymidine kinase (HSV-tk) that gains functionality by trans-splicing onto HIV-1 tat pre-mRNA. HSV-tk activates the exogenous prodrug ganciclovir for selective killing of HIV-1-infected cells. Following proof-of-principle transfection studies, therapeutic constructs were engineeredfor lentivirus-mediated delivery to HIV-infected cells in vitro. We optimized lentiviral production for high infectious titer (>106 TU/mL) and low vector plasmid carryover (<1 copy/cell). In a tissue culture model of HIV-1 infection we confirmed lentiviral delivery of therapeutic (and control) vectors. Successful trans-splicing of the HSV-tk RNA onto HIV-1 RNA in cells co-transduced with HIV-1 and therapeutic vector at an MOI as low as 4 was confirmed by sequencing. In MTT assays the most potent therapeutic vector killed approximately 80% of HIV-1-expressing cells. Next-generation vectors are being evaluated for enhanced therapeutic potential and improved HIV-1-targeting RNA expression. The lead candidate culminating from this work will be assessed for selective killing of HIV-1-infected cells both productively infected and with virus reactivated from latency.

Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression

Lentiviral vectors (LVs) are widely used for delivering foreign genes for long-term expression. Recently, virus-like particles (VLPs) were developed for mRNA or ribonucleoprotein (RNP) delivery for short-term endonuclease expression. Generating large amount of LVs or VLPs is challenging. On the other hand, methods for using VLPs to co-deliver Cas9 mRNA and single guide RNA (sgRNA) are limited. Fusing aptamer-binding protein (ABP) to the N-terminus of HIV Gag protein is currently the successful way to develop hybrid particles for co-delivering Cas9 mRNA and sgRNA. The effects of modifying Gag protein this way on particle assembly are unknown. Previously we found that adding an ABP after the second zinc finger domain of nucleocapsid (NC) protein had minimal effects on particle assembly. Based on these observations, here we developed hybrid particles for Cas9 mRNA and sgRNA co-delivery with normal capsid assembly efficiency. We further improved LVs for integrated gene expression by including an aptamer sequence in lentiviral genomic RNA, which improved lentiviral particle production and enhanced LV genomic RNA packaging. In summary, here we describe the development of new all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA, and new LVs for enhanced vector production and gene expression.

Clinical grade lentiviral vector purification and quality control requirements.

Lentiviral vectors have been proven to be a powerful tool in gene therapies that includes the ability to perform long-term gene editing in both dividing and non-dividing cells. In order to meet the rising demand for clinical-grade lentiviral vectors for future clinical trials and requirements by regulatory agencies, new methods and technologies were developed, including the rapid optimization of production and purification processes. However, gaps still exist in achieving ideal yields and recovery rates in large-scale manufacturing process steps. The downstream purification process is a critical step required to obtain a sufficient quantity and high-quality lentiviral vectors products, which is challenged by the low stability of the lentiviral vector particles and large production volumes associated with the manufacturing process. This review summarizes the most recent and promising technologies and enhancements used in the large-scale purification process step of lentiviral vector manufacturing and aims to provide a significant contribution towards the achievement of providing sufficient quantity and quality of lentiviral vectors in scalable processes.