2021 Gene and Cell Therapy Calendar - Vigene Biosciences
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V igene Biosciences, an award-winning world leader in plasmid and viral vector development and manufacturing, wishes you a healthy and prosperous 2021. This 2021 calendar contains figures and tables that have been carefully selected by Vigene from gene and cell therapy articles, all reprinted with permission from Springer Nature. We hope that it will be a useful reference for researchers in the gene and cell therapy field. Vigene Bioscience has been ranked by Inc. Magazine as one of the fastest growing companies in the USA for the past three years. Vigene offers integrated plasmid and viral vector production and analytical services from its 71,000 sq ft state-of-the-art facility which includes 10 Good Manufacturing Practice (GMP) clean-room suites. Vigene’s mission is to make gene therapy affordable. On the basic research side, Vigene is developing, manufacturing, and distributing adeno-associated virus (AAV), lentivirus, retrovirus, adenovirus, and plasmid-based reagents including Howard Hughes Medical Institute (HHMI)/ Janelia Research Campus AAV Biosensors. On the cGMP clinical production side, Vigene combines proven production technologies with rigorous, regulatory compliant cGMP production processes to meet the needs and expectations of clinical and commercial clients. Vigene offers FDA and EMA compliant cGMP production for AAV, lentivirus, adenovirus, retrovirus, and plasmids to global pharmaceutical and biotech companies, government agencies, and non-profit organizations.
Viral Vector Genome Size Infec on Expression Pote a ons onal mutagenesis Retrovirus 7-11 kb (ssRNA) Dividing cells Stable poten al Dividing & non- onal mutagenesis Len virus 9 kb (ssRNA) Stable dividing cells poten al Dividing & non- Adenovirus 36 kb (dsDNA) Transient Immune response dividing cells Adeno- Dividing & non- associated 4.7 kb (ssDNA) long las ng Immune response dividing cells Virus, AAV Herpes Simplex Dividing & non- No gene expression 150 kb (dsDNA) Transient Virus, HSV dividing cells during latent infec on Pote al cytopathic Vaccinia virus 190 kb (dsDNA) Dividing cells Transient effects Vigene integrated vector and plasmid service — Research, preclinical and clinical Vigene is a world leader in plasmid and viral vector development and manufacturing. Vigene has expertise in development, manufacturing and analytics for plasmid vectors and many viral vectors, including AAV, lentivirus, adenovirus, retrovirus, HSV, and vaccinia virus. Please see the Jan 2021 promotion here: vigenebio.com/2021/Jan
Vigene integrated vector and plasmid service MONDAY TUESDAY WEDNESDAY THURSDAY JANUARY FRIDAY 2021 SATURDAY SUNDAY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 www.vigenebio.com
Transmission electron microscopy of rAAV. It is clear that full particles (open arrow) stain differently from empty particles (darkly stained center; small dark arrow). Viewing numerous fields similar to this will allow determination of the full-to-empty particle ratio. (Figure 8, Nature Protocols 1, 1412–1428 (2006)) Figure reprinted with permission from Springer Nature AAV reference standards The gene and cell therapy field needs good AAV reference standards. Pursuant to the Jan 2020 FDA Gene Therapy Chemistry Manufacturing and Controls (CMC) guidance, Vigene’s AAV reference standards are as follows: • AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 serotypes • Full and empty capsids • AAV titer standards Please view the details and promotions at: vigenebio.com/2021/Feb
AAV reference standards MONDAY TUESDAY WEDNESDAY FEBRUARY THURSDAY FRIDAY 2021 SATURDAY SUNDAY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 www.vigenebio.com
ITR ITR AAV vector packaging (4.5-kb capacity without engineering) Promoter Therapeutic transgene Poly(A) 5′ 3′ OH Recombinant AAV virion • Screen for and Use of dual vectors that take • Mutation of ITRs • Codon optimization Engineering use small, strong, advantage of: to generate self- via codon usage bias • Synthetic poly(A) approach tissue-specific • AAV genome concatemerization complementary • Interference of • Reversed poly(A) promoters and • Homologous recombination AAV intermediates antigen presentation enhancer elements • A hybrid dual-vector strategy • Intein-mediated protein • Increases • Enhances trans-splicing technology tissue-specific polyadenylation Cross-packaging of an AAV • Omits need transcription • Increases • Minimizes kilobases of genome Positive for second- of transgene transcription DNA taken up by poly(A) effect on strand synthesis • Minimizes and translation of • Avoids rolling circle transduction of ssDNA kilobases of DNA the transgene transcription and Pol II taken up by jumping (between • Increases size of transgene that promoter concatamers) can be packaged • Decreases innate Positive • Decreases immune response • Decreases innate effect on • Decreases CTL innate immune to AAV immune response immune response to AAV response to AAV • Reduces CTL to AAV response response Engineering the AAV cassette. The adeno-associated virus (AAV) cassette can be engineered to enhance AAV transduction and also to enable AAV to escape immune responses. Mutation of one inverted terminal repeat (ITR) on the AAV vector, which prevents the nicking of Rep protein, can generate self-complementary AAV vector to enhance vector transduction. Mutation of ITRs may also decrease the innate response to AAV. Use of small tissue-specific promoters increases tissue-specific transgene expression and the packaging capacity of the AAV genome and minimizes the cytotoxic T lymphocyte (CTL) immune response to AAV. Optimization of transgene codons increases the transcription and translation of the AAV transgene and decreases the immune response to AAV. Using synthetic poly(A) can increase the nuclear export, translation and stability of mRNA (by enhancing polyadenylation), and using reversed poly(A) can avoid the transcription of ITR; both approaches enhance AAV transduction, and reversed poly(A) decreases the innate response to AAV. Finally, the use of dual AAV vectors or the cross-packaging of the AAV genome enables effective and functional expression of large transgenes. Pol II, DNA polymerase II; ssDNA, single-stranded DNA. (Figure 2, Nature Reviews Genetics 21, 255–272 (2020)) Figure reprinted with permission from Springer Nature Plasmid production – Research, preclinical, clinical • Research grade & GMP-ReadyTM, GMP production • Ready-to-use, GMP-Ready pHelper & other viral vector packaging plasmids • Endotoxin free & supercoil plasmid homogeneity • Animal component free production process Please view the details at vigenebio.com/2021/Mar
Plasmid production – preclinical and GMP MONDAY TUESDAY WEDNESDAY THURSDAY MARCH FRIDAY 2021 SATURDAY SUNDAY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 www.vigenebio.com
TABLE 1: AAV PRODUCTION OPTIONS Pros and cons of various recombinant adeno-associated virus (rAAV) manufacturing strategies. rAAV cannot replicate without a helper virus, and early manufacturing efforts entailed coinfection of host cells with adenovirus or herpesvirus. Newer strategies replace these with plasmids containing key helper virus genes, or combine all necessary genetic elements into an insect cell-specific baculovirus vector. Production is most efficient in free-floating suspension cells, but substrate-attached adherent cell lines can also achieve reasonable viral output. AAV MANUFACTURING KEY KEY PRODUCTION CELL LINE CHOICES TECHNOLOGY STRENGTHS DRAWBACKS ADHERENT SUSPENSION • Helper virus • Highly scalable contamination • Serum-free media • Long lead time for cell HEK293/293T HEK293/293T-s Helper virus • Efficient line and virus seed production in generation HeLa HeLa-s suspension culture • May require serum- containing media • May require serum- • No helper virus containing media contamination • Large proportion of Helper-free triple • Rapidly produce empty capsids HEK293/293T HEK293/293T-s transfection virus in small scale • Supply of plasmids for • Simple procedure large-scale production can be costly • Baculovirus virus • Highly scalable contamination Serum-free media • Baculovirus instability Baculovirus • Efficient — sf9 production in • Long lead time for cell suspension culture line and virus seed generation AAV production – Research, preclinical, clinical • Research grade, preclinical, clinical, and commercial GMP AAV production • Proprietary cGMP released MCB of HEK293, 293T adherent and suspension cell lines • Established and proven high productivity AAV production process • >15,000 research batches, >50 preclinical and clinical batches released Please view the promo at vigenebio.com/2021/Apr
AAV production – preclinical and GMP MONDAY TUESDAY WEDNESDAY THURSDAY APRIL FRIDAY 2021 SATURDAY SUNDAY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 www.vigenebio.com
Target cell Virus-mediated transduction is a useful tool to generate various types of next-generation stem cells Virus as a key tool Click chemistry Nuclease or AAV-based gene editing a Prodrug-converting enzymes b Optogenetic actuator-embedded c CAR-expressing HSCs or or oncolytic virus delivery via stem cell derivative PSC-derived NK cells tumour-homing NSCs/MSCs CAR Stem cell Oncolytic virus particles (or prodrug- converting enzymes) e Cytokine/growth factor-overexpressing stem cells to enhance endogenous repair d Gene therapy of stem cells to provide long-term functional compensation for disease-causing, inherited mutation f Improved delivery of anticancer g Gene editing tools can be used to knock agents to sites of action using click out expression of HLA genes to reduce the chemistry to tether them to stem cells immunogenicity of allogeneic stem cells Azide DBCO HSC Platelet Anti-PD-L1 The stem cell toolkit and its application in developing next-generation stem cells. Virus-mediated transduction of stem cells (target cell), particularly with self-inactivating lentiviruses, is a useful tool for the creation/development of next-generation stem cells. Viruses can be used to engineer: prodrug-converting enzymes, oncolytic viruses and other anticancer drugs into neural stem cells (NSCs) and mesenchymal stem cells (MSCs) (part a); optogenetically enhanced stem cell derivatives to provide light-inducible control over the activity of trans- planted stem cells/progenitors (part b); chimeric antigen receptor (CAR)-expressing haematopoietic stem cells (HSCs) and pluripotent stem cell (PSC)-derived natural killer (NK) cells for immune-oncology applications (part c); gene therapy in HSC, skin and muscle progenitors to treat inherited diseases (part d); and cytokine/growth factor delivery in MSCs/neural progenitors to stimulate endogenous tissue repair (part e). Click chemistry is another engineering tool that can be used to tether anticancer agents to stem cells for improved delivery to hard-to-reach cancers, such as leukaemia cells residing deep in the bone marrow (part f). Advances in gene editing technology have made this a versatile tool to precisely edit specific loci within the genome, and gene editing technology has become the tool of choice for the creation/development of universally immunocompatible PSC lines and derivatives (part g). Several of these next-generation stem cell-based therapies have already reached clinical testing, whereas others in preclinical development are not far behind. AAV, adeno-associated virus; DBCO, dibenzocyclooctyne; HLA, human leukocyte antigen; PD-L1, programmed cell death protein 1 ligand. (Figure 1, Nature Reviews Drug Discovery 19, 463–479 (2020)) Figure reprinted with permission from Springer Nature Lentivirus production – Research, preclinical, clinical • Research grade, preclinical, clinical, and commercial GMP lentivirus production • High titer, purified, ready to use in vitro and animal studies — research grade • Chromatography based commercial ready lenti production — GMP • High transduction efficiency Please view the promo at vigenebio.com/2021/May
Lentivirus research production MONDAY TUESDAY WEDNESDAY THURSDAY MAY FRIDAY 2021 SATURDAY SUNDAY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 www.vigenebio.com
Oncolytic Cytokine virus receptors Release/ secrete Infection Cancer cell NK cell Cytotoxicity • Viral proteins • Viral genome CD8+ T cell • ER stress ROS Cytokine • Genotoxic stress receptors IL-2R Cytotoxicity IL-2 Viral oncolysis ROS Release Release CD28 TCR CD4+ T cell Antigen uptake MHC MHC CD40L TLR PAMPs DAMPs Cytokines • Viral capsids • HSPs • Type I interferons • Viral DNA • HMGB1 • TNFα Activation CD40 • Viral dsRNA/ssRNA • Calreticulin • IFNγ TCR • Viral proteins • ATP • IL-12 • Uric acid MHC • Type I IFNs • Type I IFNs DAMPs/ • DAMPs/PAMPs • Cytokines PAMPs • Viral antigens • CD80/CD86 • TAAs/neoantigens • Chemokine receptors Antigen presenting cell The induction of local and systemic anti-tumour immunity by oncolytic viruses. The therapeutic efficacy of oncolytic viruses is determined by a combination of direct cancer cell lysis and indirect activation of anti-tumour immune responses. Upon infection with an oncolytic virus, cancer cells initiate an antiviral response that consists of endoplasmic reticulum (ER) and genotoxic stress. This response leads to the upregulation of reactive oxygen species (ROS) and the initiation of antiviral cytokine production. ROS and cytokines, specifically type I interferons (IFNs), are released from the infected cancer cell and stimulate immune cells (antigen presenting cells, CD8+ T cells, and natural killer (NK) cells). Subsequently, the oncolytic virus causes oncolysis, which releases viral progeny, pathogen-associated molecular patterns (PAMPs), danger-associated molecular pattern signals (DAMPs), and tumour associated antigens (TAAs) including neo-antigens. The release of viral progeny propagates the infection with the oncolytic virus. The PAMPs (consisting of viral particles) and DAMPs (comprising host cell proteins) stimulate the immune system by triggering activating receptors such as Toll-like receptors (TLRs). In the context of the resulting immune-stimulatory environment, TAAs and neo-antigens are released and taken up by antigen presenting cells. Collectively, these events result in the generation of immune responses against virally infected cancer cells, as well as de novo immune responses against TAAs/neo-antigens displayed on un-infected cancer cells. CD40L, CD40 ligand; dsRNA, double-stranded RNA; HMGB1, high mobility group box 1; HSP, heat shock protein; IL-2, interleukin-2; IL-2R, IL-2 receptor; MHC, major histocompatibility complex; ssRNA, single-stranded RNA; TCR, T cell receptor; TNFα, tumour necrosis factor-α. (Figure 2, Nature Reviews Drug Discovery 14, 642–662 (2015)) Figure reprinted with permission from Springer Nature Adenovirus production - Research, preclinical, clinical • Research grade, preclinical, clinical, and commercial GMP adenovirus production • High titer, purified, ready to use in vitro and animal studies – research grade • Chromatography based commercial ready adenovirus production – GMP • Oncolytic virus and vaccine production process ready Please view the promo at vigenebio.com/2021/June
Adenovirus production MONDAY TUESDAY WEDNESDAY THURSDAY JUNE FRIDAY 2021 SATURDAY SUNDAY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 www.vigenebio.com
Table 1 | Properties Properties and clinical anduse of AAV clinical use serotypes of AAV serotypes AAV Origin of Primary Co-receptor Tissue tropism Condition Approved drug serotype isolation receptor (ClinicalTrials.gov identifier) AAV1 Monkey Sialic acid AAVR Muscle, CNS, heart Muscle diseases (NCT01519349) None Heart failure (NCT01643330) AAT deficiency (NCT01054339, NCT00430768) AAV2 Human Heparin Integrin, FGFR , HGFR, Liver, CNS, muscle Eye diseases (NCT00643747) Luxturna for Leber LamR, AAVR congenital amaurosis Haemophilia (NCT00515710) CNS diseases (NCT00400634) AAT deficiency (NCT00377416) AAV3 Human Heparin FGFR , HGFR LamR, Muscle, stem cells No trials underway None AAVR AAV4 Monkey Sialic acid Unknown Eye, CNS Eye diseases (NCT01496040) None AAV5 Human Sialic acid PDGFR, AAVR CNS, lung, eye Haemophilia (NCT03520712) None Eye diseases (NCT02781480) AIP (NCT02082860) AAV6 Human Heparin, EGFR , AAVR Muscle, CNS, heart, Haemophilia (NCT03061201) None sialic acid lung CNS diseases (NCT02702115) AAV7 Monkey Unknown Unknown Muscle, CNS No trials underway None AAV8 Monkey Unknown LamR, AAVR Liver, muscle, Eye diseases (NCT03066258) None pancreas, CNS Haemophilia (NCT00979238) Muscle diseases (NCT03199469) AAV9 Human Galactose LamR, AAVR Every tissue CNS diseases (NCT02122952) Zolgensma for spinal muscular atrophy Muscle diseases (NCT03362502) AAV10 Monkey Unknown Unknown Muscle No trials underway None AAV11 Monkey Unknown Unknown Unknown No trials underway None AAV12 Human Unknown Unknown Nasal No trials underway None AAT,α1-antitrypsin; AAV, adeno-associated virus; AAVR, AAV receptor; AIP, acute intermittent porphyria; CNS, central nervous system; EGFR, epidermal growth factor AAT, α1-antitrypsin; AAV, adeno-associated virus; AAVR, AAV receptor; AIP, acute intermittent porphyria; CNS, central nervous system; EGFR, receptor ; FGFR , fibroblast growth factor receptor ; HGFR, hepatocyte growth factor receptor ; LamR, laminin receptor 1; PDGFR , platelet-derived growth factor receptor. epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; HGFR, hepatocyte growth factor receptor; LamR, laminin receptor 1; PDGFR, platelet-derived growth factor receptor. (Table 1, Nature Reviews Genetics 21, 255–272 (2020)) Table reprinted with permission from Springer Nature AAV, lentivirus and adenovirus controls • Reporters available: RFP, GFP, mCherry, Luciferase, Cre, LacZ • Promoters available: ALB, aMHC, c-Fos, CAG, CaMKIIa, CK0.4, CK1.3, CMV, cTnT, EF1a, EFFS, GFAP, HCRApoE, MBP, MCK, MeCP2, NSE, PDX1, PGK, Rpe65, SST, Syn, TBG, UBC, DIO-GFP, DIO-mCherry, DIO-RFP, DIO-LacZ Please view the promo at vigenebio.com/2021/July
AAV controls and lentivirus control MONDAY TUESDAY WEDNESDAY THURSDAY FRIDAY JULY 2021 SATURDAY SUNDAY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 www.vigenebio.com
Table 1 Example Example of characterization testing of characterization for for testing an an HEK293 HEK293master master cell bank cell bank Test M et hod S pecifi cat ion Microbial Fo r m icr o b ia l co n t a m in a t io n Tr a n s m is s io n e le ct r o n m icr o s co p y No viruses, virus-like particles, mycoplasmas, fungi, yeasts, bacteria Bacteriostatic/fungistatic activity of test article Four media, direct inoculation No bacterial and fungal activity Bacterial and fungal contaminants Four media, direct inoculation Negative Agar cultivable and noncultivable mycoplasmas 1993 points to consider Negative General viruses Inapparent viruses In vivo Not detected Viral contaminants In vitro assay for the presence of Not detected viral contaminants Specific human viruses CM V PCR N e g a t iv e EBV PCR N e g a t iv e H AV RT-P C R N e g a t iv e H BV PCR N e g a t iv e H CV RT-P C R N e g a t iv e H H V-6 PCR N e g a t iv e H H V-7 PCR N e g a t iv e H H V-8 PCR N e g a t iv e H IV-1/ 2 PCR N e g a t iv e H u m a n p a r v o v ir u s B19 PCR N e g a t iv e H TLV I/ II PCR N e g a t iv e Re t r o v ir u s e s Q -P ERT < 5.0 10 7 U ml 1 Specific simian viruses SFV PCR N e g a t iv e SRV RT-P C R N e g a t iv e STLV PCR N e g a t iv e SV40 PCR N e g a t iv e Other specific viruses Bovine viruses In vitro assay for the presence of Not detected bovine viruses (9CFR) Porcine viruses In vitro assay for the presence of Not detected porcine viruses (9CFR) Identity C e ll id e n t ifi ca t io n Is o e n z y m e a n a ly s is Is o e n z y m e m i g r a t i o n d i s t a n c e s c o n s i s t e n t with cells of human origin Abbreviations: CFR, Code Abbreviations: CFR, of Federal Code of Regulations;CMV, Federal Regulations; CMV,cytomegalovirus; cytomegalovirus; EBV, EBV, Epstein–Barr Epstein–Barr virus; virus; HAV,HAV, hepatitis hepatitis A virus; A virus; HBV,HBV, hepatitis hepatitis B B virus; HCV, virus;hepatitis C virus; HCV, hepatitis HHV,HHV, C virus; human herpes human virus; herpes HIV-1/2, virus; human HIV-1/2, immunodeficiency human immunodeficiency virus virus types types 11 and 2; HTLV and 2; HTLV I/II, I/II, human T-cell lymphotropic human T-cell virus types I and lymphotropic virusII;types SFV,Isimian foamy and II; SFV, virus;foamy simian SRV, virus; simian retroviruses; SRV, STLV, simian simian retroviruses; STLV,T-lymphotropic virus; virus; simian T-lymphotropic SV40,SV40, simian virusvirus simian 40. 40. US Dept Health US DeptServices, Human Health Human Food andServices, Food and Drug Administration. Drug Administration 23 23 . (Table 1, Gene Therapy 15, 840–848 (2008)) Table reprinted with permission from Springer Nature Quality control analytical services • Plasmid QC ◆ Purity & identity: % supercoil (HPLC), sequencing, residual host DNA/RNA/proteins ◆ Safety: sterility, endotoxin, mycoplasma, etc. • Viral vector QC ◆ Purity and impurities: residual IDX (HPLC), residual host DNA/RNA/proteins ◆ Strength & safety: vector genome titer (qPCR, ddPCR), total particle titer (ELISA), sterility, mycoplasma, etc. Please view the promo at vigenebio.com/2021/Aug
Viral vector and plasmid analytical services MONDAY TUESDAY WEDNESDAY THURSDAY AUGUST FRIDAY 2021 SATURDAY SUNDAY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 www.vigenebio.com
Selected Table BCMABCMA 1 Selected CAR-T trialstrials. CAR-T Institution/ Vector/co-stimulatory domain BCMA positivity requirement No of patients Median prior lines Efficacy Safety developer (range) ORR*/PFS CRS/ICANS (months) NCI γ-retrovirus/CD28 >50% 24 9 (3–13) 81%/7.2 94% (38% grade ≥ 3)/NE UPenn Lentivirus/4–1BB Not required 25 7 (3–13) Cohort 1 44%/2.2 88% (32% grade ≥ 3)/32% Cohort 2 20%/1.9 (12% grade ≥ 3) Cohort 3 64%/4.2 Bb2121 Lentivirus/4-1BB ≥50% in dose escalation, NR in 33 7 (3–14) 85%/11.8 70% (6% grade ≥ 3)/42% dose expansion 8 (3–23) (3.3% grade ≥ 3) Bb21217 Lentivirus/4-1BB; PI3K inh during in vivo >50% 22 7 (4–17) 83%/NR 59% (4.5% grade ≥ 3)/22% expansion (9% grade≥3) LCAR-B38 M Lentivirus/41-BB Required 57 3 (1–9) 88%/15 90% (grade 3 ≥ 7%)/2% LCAR-B38M Lentivirus/4-1BB Required 17 4 (3–11) 88%/NR 100% (grade ≥ 3 35%)/NR Poseida (P-BCMA 101) PiggyBAC/4-1BB Not required 23 6 (3–11) 63%/NR 9.5%/4.8% (grade ≥ 3 4.8%) JCARH125 Lentivirus/4-1BB Not required 44 7 (3–23) 82%/NR 80% (9% grade ≥ 3)/25% (grade ≥ 3 7%) MCARH171 Retrovirus/4-1BB/tEGFR Required 11 6 (4–14) 64%/NR 60% (20% grade ≥ 3)/NR Han et al Lentivirus/4-1BB/Alpaca VHH Not required 16 10 (NR) 100%/NR NR (12.5% grade ≥ 3)/NR FCARH143 Lentivirus/4-1BB/tEGFR ≥5% 7 8 (6–11) 100%/NR 86%/0% CARTITUDE-1 Lentivirus/4-1BB Not required 25 5 (3–16) 91%/NR 80% (8% grade ≥ 3)/12% (4% grade ≥ 3) CT053 Lentivirus/4-1BB ≥50% 16 NR 100%/NR 18% (6% grade ≥ 3)/NR CT103 Lentivirus/4-1BB NR 16 4 (3–5) 100%/NR 100% (37.5% grade ≥ 3)/0% Cowan et al Lentivirus/4-1BB Required 8 10 (4–23) 100%/NR 100%/70% GSI (JSMD194)/tEGFR C-CAR088 Lentivirus/4-1BB NR 3 7 (NR) 100%/NR NR HRAIN biotechnology γ-retrovirus-4-1BB/tEGFR >5% 17 NR 79%/NR NR CRS cytokine release syndrome, ICANS immune effector cell associated neurotoxicity syndrome, ORR overall response rate, PFS Progression free survival, BCMA B cell maturation antigen, GSI CRS cytokine release syndrome, ICANS immune effector cell associated neurotoxicity syndrome, ORR overall response rate, PFS Progression free survival, BCMA B cell gamma secretase inhibitor, EGFR epidermal growth factor receptor. maturation antigen, GSI gamma secretase inhibitor, EGFR epidermal growth factor receptor. *responses *responsesassessed afterafter assessed 30 days. 30days. (Table 1, Bone Marrow Transplantation https://dx.doi.org/10.1038/s41409-020-01023-w (2020)) Table reprinted with permission from Springer Nature B. Dhakal et al. Retrovirus production – Research, preclinical, clinical • Research grade, preclinical, clinical, and commercial GMP retrovirus production • High titer, purified, ready to use in vitro and animal studies – research grade • Chromatography based commercial ready retrovirus production – GMP • High transduction efficiency Please view the promo at vigenebio.com/2021/Sept
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Conventional ssAAV ScAAV wtTR ΔTR 2.3 kb 4.6 kb wtTR wtTR 4.6 kb 4.6 kb 3ʹ 5ʹ 3ʹ 5ʹ + Open Open + 4.6 kb 4.6 kb 3ʹ 5ʹ 3ʹ 5ʹ – Open Open – Graphical representation of the suggested portion of the transgene plasmid to be used as the template of the probe for dot-blot analysis. The black lines represent sequences in the bacterial backbone; the blue and red lines represent sequences of the coding and complementary sequence of the transgene expression cassette, respectively. (Figure 7, Nature Protocols 1, 1412–1428 (2006)) Figure reprinted with permission from Springer Nature AAV & LVV plasmid ITR/LTR repair and production service Plasmid design and cloning (identical ITR vs hybrid) • Genetic stability study & ITR sequencing analysis with NGS • AAV plasmid & LVV plasmid clonal screening using different E. coli strains for stable and reproducible ITR/LTR plasmid production Please view the promo at vigenebio.com/2021/Oct
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Packaging and payload Delivery Tropism Local Global injection injection All cells Selected cells Gene X Virus particle Infected cell Uninfected cell Genetic access Infectivity and toxicity Transgene expression Entry Virus Transgene expression (susceptibility) Infectivity injection Healthy cell m7G AAA Transgene expression (permissivity) Toxicity Damaged Duration of Decay or cell expression toxicity Onset Key principles for viral-mediated gene transfer in neuroscience. Schematic demonstrating six key principles essential for the neuroscientist: viral packaging limit (how much nucleic acid a virus particle can carry) and payload (the length and type of genomic material that can be successfully packaged into a virus particle), delivery methods (local versus global injections), tropism (specificity of a virus for a given cell type(s)), access (ability of a virus to enter a cell type and express its gene product(s)), infectivity and toxicity (how efficiently a virus infects a cell and how harmful it is to the cell), and transgene expression dynamics (time course of onset and persistence of transgene expression). These principles play a key role in determining a neuroscientist’s choice of virus by weighing the advantages and disadvantages of a given virus. AAA, 3´ poly(A) tail for mRNA; Gene X, a transgene being packaged into a virus particle; m7G, 7-methylguanosine (5´ cap for mRNA). (Figure 1, Nature Reviews Neuroscience 21, 669–681(2020)) Figure reprinted with permission from Springer Nature Gene therapy bioinformatics & resources central • Technology handbooks • Bioinformatics tools • Webinar tutorials Please view the promo at vigenebio.com/2021/Nov
Gene therapy tools MONDAY TUESDAY WEDNESDAY NOVEMBER THURSDAY FRIDAY 2021 SATURDAY SUNDAY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 www.vigenebio.com
a b c d 2D cell factories Microcarrier culture (1) Subculture — Modified surfaces bead-to-bead transfer (2) Dissociation Solid microcarrier Degradable Enzymatic surface digestion Engineered substrates PNIPAM n O N T < 32 °C T > 32 °C Centrifugal separation Bottlenecks Solutions Process optimization for the expansion of cells and for cell collection from microcarriers. a, Production of clinical lots by using adherent MSCs in 2D cell-culture plates. Issues with the scaling of costs and labour efficiency make 2D culture unlikely to meet an estimated demand of > 1012 viable cells per year, necessary for treating prevalent adult indications. b, Suspension culture systems for MSCs use microcarriers and stirred tank bioreactors and are a scalable and sustainable approach for cell expansion at high density. c, Unit operations identified as major bioprocessing bottlenecks: (1) bead-to-bead transfer for MSC subculturing and expansion; (2) the need for enzymatic digestion and centrifugal separation to isolate the MSCs from the microcarriers. d, Materials-science innovations in microcarrier substrates can improve product purity, identity and potency through degradable and temperature (T)-sensitive materials (such as poly(N-isopropylacrylamide), PNIPAM) that remove the need for additional enzymatic dissociation processes. (Figure 1, Nature Biomedical Engineering 2, 362–376 (2018)) Figure reprinted with permission from Springer Nature Bioprocess and GMP production • FDA/EMA compliant with 17-yr GMP production records • Established AAV, LVV, adenovirus, retrovirus, and plasmid GMP production processes • >10 GMP suites, >100,000 sq ft GMP & lab space for adherent and suspension cell production (50 L to 1,000 L per batch) Please view the promo at vigenebio.com/2021/Dec
Gene therapy tools MONDAY TUESDAY WEDNESDAY DECEMBER THURSDAY FRIDAY 2021 SATURDAY SUNDAY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 www.vigenebio.com
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