AAV, Adenovirus and Lentivirus-based Gene Delivery Handbook

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1 CMYK C: 73% M:5% Y:31% K:0% C: 49% M:41% Y:41% K:5% Pantone Coated Pantone 319C Pantone Cool Gray 9C AAV, Adenovirus and Lentivirus-based Gene Delivery Handbook MP With cg T Rand CA section

2 Table of Contents Introduction Viral vector selection guide How does virus-based gene delivery work?... 4 AAV overview and serotype selection guide... 5 AAV advantages, packaging and transduction... 6 AAV titer and purity AAV expression data Adenovirus overview Adenovirus FAQs and caveats Lentivirus overview Lentivirus cgmp production for viral vectors Tissue specific promoter list for viral vectors Vigene product and service guide Vigene Biosciences

3 Introduction The development of viral gene delivery systems including lentivirus, adenovirus, and adenoassociated virus (AAV) has empowered biomedical researchers to introduce genes into diverse types of cells and tissues, including those which are hard to transfect. Selecting an appropriate viral vector requires consideration of many factors such as the target cell type and the size of the genetic cargo. The table below summarizes the main factors to consider in selecting a viral system for gene delivery. Adeno-associated Virus (AAV) Adenovirus Lentivirus Genome ssdna dsdna ssrna (+) Coat Naked Naked Enveloped Genome size 5kb 38-39kb 9kb Infection/tropism Dividing and nondividing cells Dividing and nondividing cells Dividing and nondividing cells Host Genome Interaction Transgene expression Packaging Capacity Immune Response Relative Viral Titer Relative Transduction Efficiency Non-integrating Non-integrating Integrating Potentially long-lasting Transient Long-lasting 4.5kb 7.5kb 6kb Very Low High Low 10^7 without concentration 10^11 without purification 70% 100% 70% 10^7 without concentration Relative Foreign Medium High Medium Gene Expression More details AAV packaging service Adenoviral packaging service Lentiviral packaging service AAV, Adenovirus and Lentivirus-based Gene Delivery Handbook 3

4 How does virus-based gene delivery work? Viruses can infect cells and hijack the gene expression machinery of their hosts to express viral genes. Typically a viral particle enters a host cell via clathrin-mediated endocytosis. Then the viral nucleic acid is released into to the host cell where it may remain episomal or integrate into the host genome, depending on the virus type. Recombinant AAVs (raav) used in research laboratories typically do not integrate their nucleic acid sequences into the host genomes. Wild-type AAV is unique among mammalian viruses in that it demonstrates sitespecific integration into the host genome at the AAVS1 loci of human chromosome 19, but raav does not have this capability; instead, raav genomes are maintained as episomes, with only infrequent integration into the host genome at random sites. In order to create new viral particles, AAV requires co-infection with a helper virus such as adenovirus. Adenoviruses do not integrate their nucleic acid into their host genomes; they exist in episomal format. Gene expression, using both cellular and viral proteins, is initiated to systematically produce the proteins necessary to build new virion particles. Lentiviruses use viral reverse transcriptase to create DNA copies of their RNA genome, and cellular machinery to create the second strand. The resulting double-stranded molecule can integrate anywhere within the mammalian genome using a virally encoded integrase. All three viral delivery methods are used as tools for biomedical research and as therapeutic reagents in cell and gene therapies. 4 Vigene Biosciences

5 Adenovirus Associated Virus (AAV) and serotype selection Adeno-associated virus (AAV) is a small single-stranded DNA virus that infects cells of humans and some other primate species. AAV is not currently known to cause disease and generates only a very mild immune response. It can infect dividing and non-dividing cells. Those features make raav an ideal viral vector for gene therapy. AAVs can be used to efficiently deliver genes into many types of tissue. Distinct AAV serotypes demonstrate tissue tropism that is, specificity in the types of tissues they infect due to variations in the capsid protein. So far there are 11 AAV serotypes described, and efforts are underway to engineer novel tissue-specific serotypes. Selecting the right serotypes is critical to the success of gene delivery experiments. The following table lists the most popular raav serotype and their tropism. AAV Serotype Muscle hepatocyte pulmonary neurons & glial cells retinal pigmented epithelium pancreas kidney AAV1 neurons & glial cells AAV2 AAV5 lung alveolar cells neurons & glial cells AAV6 AAV7 neurons AAV8 neurons AAV9 AAV, Adenovirus and Lentivirus-based Gene Delivery Handbook 5

6 AAV advantages, packaging and transduction Benefits Ideal for in vivo application No/little immune response for in vivo applications and gene therapy Optimal diffusion when used in vivo Non-integrative transduction genetic footprint free cell engineering Tissue specific targeting by serotypes Caveats Moderate expression level in vitro Not integrative not useful for making stable cell lines. Vigene Biosciences utilizes the 3-plasmid system for raav production. AAV virus packaging overview Compared to wild-type AAV, raav has two essential genes, rep and cap, replaced with the transgene cassette insertion site. Generating raav particles requires transfecting producer cells (typically HEK293) with the following 3 components: raav cis-plasmid containing the transgene expression cassette (maximum of 4.9 kb) flanked by the 145 nucleotide-long AAV ITRs raav helper plasmid containing rep and cap genes to express these proteins in trans A plasmid containing the adenovirus helper factors E2A, E4ORF6 and virus-associated RNAs (VA RNAs). [Another required factor, the E1A/E1b gene, is already expressed in HEK293 cells so does not need to be included in this plasmid.] AAV transduction overview AAV infection is cell type dependent. Some cell types exhibit low transduction efficiency, while others transduce very readily. When designing AAV transduction experiments, it is recommended to test several different serotypes and to use a reporter vector (such an AAV expressing GFP) to determine the optimal serotype for transduction of the desired tissue or cell culture. Start transducing the cells at a multiplicity of infection (MOI) between 1x10^4 and 1x10^6 viral genomes per cell (vg/cell) if the cells are readily transducible. With some cell lines a higher MOI might be needed. Look for the highest transduction with minimal cell death. With some cell lines, high transduction levels cannot be achieved. pav-fh AAV Vector Transfection into HEK293 Cell HEK293 Cell AAV Virus Particles Ad Helper Vector AAV Rep/Cap Vector GFP Expression (Green) in neuronal cells in the spinal chord & brain, 4 weeks after injection of raav GFP vector. Courtesy of Dr. Wang Chen, Harvard University 6 Vigene Biosciences

7 Titer and purity of AAV Titer and purity are two important factors that determine the efficacy and safety of AAV. Effective gene delivery depends on using the optimal multiplicity of infection (MOI), and knowing the titer of the virus is essential to using the correct MOI. There are two main types of viral titer: 1. Physical titer - a measurement of how much virus is present, usually expressed as the number of viral particles per ml (VP/mL), viral genomes per ml (VG/mL) or genome copies/ml (CG/ml) 2. Functional titer, also known as infectious titer, is the measurement of how much virus actually infects a target cell. Functional titer may be expressed in the form of transduction units per ml (TU/mL), plaqueforming units per ml (pfu/ml), or infectious units per ml (ifu/ml), depending on the viral vector. Functional titer is always a more relevant measurement because it measures how much virus actually gets into the target cell. However, physical titer is much easier to obtain and takes much less time to measure. Functional titer may be estimated from physical titer; functional titer will always be lower than physical titer, usually by a factor of 10 to 100-fold. The purity of AAV preparations has important implications for both safety and efficacy of clinical gene transfer. Typically AAV purity is assessed by SDS-PAGE followed by silver staining to confirm efficient removal of protein impurities. In addition, A260/A280 spectrophotometric analysis of vector preparations can be used to provide a quantitative estimate of nucleic acid and protein impurities in various vector preparations. Benefits of high titer high purity AAV Vigene Biosciences, a leader in viral particle based gene delivery, applies proprietary technologies to achieve high titer AAV viral particles (10^13-10^14 GC/ml). This enables: Higher transduction rates More animal injections per batch Reproducible results from viral particles of the same batch Viral titer is measured by qpcr of viral particles or by qpcr of viral infection unit in HEK293 cells Purity of purified AAV virus. Samples containing 5X10^11 and 5X10^10 AAV-GFP virus particles were analyzed by SDS-PAGE and Coomassie blue staining. Only three virus proteins are in the purified virus sample. AAV, Adenovirus and Lentivirus-based Gene Delivery Handbook 7

8 AAV expression data Recombinant adeno-associated virus (raav) vectors are capable of mediating long-term gene expression as episomal monomeric and concatemeric circles. The persistence of the vector genomes and gene expression for years makes AAV an ideal vector if enduring expression of a transgene is desirable. GFP-rAAV expression in neuronal tissues cortex Spinal Cord 5 Weeks after AAV9-GFP injection in mouse. GFP signal could be detected throughout the nervous system. The strongest signal was seen in the neuronal cell body and dendritic fibers. Hippocampus AAV shrna Advantages shrna construction for lenti, adenoviral and AAV vectors Knock down up to 4 different genes using one single plasmid vector Knock down one gene with 4 different shrnas in one vector 4in1 shrna Vector 8 Vigene Biosciences

9 Adenovirus overview The adenovirus genome is a linear double-stranded (ds) DNA that is between 26 and 48 kb in size. Replication defective recombinant adenovirus is one of the most efficient gene delivery vehicles for use both in vitro and in vivo. Compared to transfection methods or to other viral delivery systems, adenovirus has a much larger transgene capacity; adenoviral vectors can accommodate cargo up to 30 kb in length, making it the ideal choice for cell therapy. The primary drawback of recombinant adenovirus for gene delivery is that it can induce severe immunogenic responses; humans carry neutralizing antibodies to adenovirus, which may cause respiratory, gastrointestinal and eye infections. Recombinant adenoviral gene delivery systems offer several benefits: High gene expression level can be achieved in a broad range of hosts, including both dividing and non-dividing mammalian cells. No host genome integration and no activation or inactivation of host gene expression. Gene expression can often be observed in less than 24 hours. Cloning into recombinant adenovirus vector is not an easy task. Traditionally, recombinant adenovirus vectors were generated through homologous recombination in 293 cells, transfected with a plasmid containing the 5 portion of the viral genome and a 3 fragment of viral DNA segments. Newer techniques, including directional cloning, have been developed to make the cloning process more efficient. Vigene Biosciences features the world s largest selection of premade adenovirus particles containing human cdna clones. 15,000 premade adenoviruses carrying full length human cdna (1 gene/adenovirus) are available immediately. AAV, Adenovirus and Lentivirus-based Gene Delivery Handbook 9

10 Adenovirus FAQs and caveats FAQ Is recombinant adenovirus safe? Adenoviral vectors used in research are replication deficient, and are therefore considered to be safe and can be handled in BSL2 labs. Premade recombinant adenovirus particles lack E1 and E3 due to gene deletion, and are therefore unable to replicate except in the packaging cell line. They should be treated like other recombinant DNA materials, for example, plasmid clones. Is gene expression from recombinant adenovirus transient or stable? When a gene is delivered into mammalian cells by recombinant adenovirus, the expression is transient. Unlike lentivirus, recombinant adenovirus genomes remain epichromosomal in host cells. Is recombinant adenoviruses toxic to host cells? Recombinant adenovirus may trigger immune responses in vivo, but it shows no toxicity to transduced cells in vitro. Posttransfection viability of the host cells is almost 100%; adenovirus is well tolerated in a wide range of cells unlike plasmid transfections in which toxic chemicals must be used. Caveats Cloning into adenoviral vectors is challenging due to the large genome size. Does not integrate into host genome; not suitable for creating stable cell lines. Vigene Biosciences guarantees that every adenovirus packaged human full-length cdna can be expressed in HEK293 cells (western blot). 42hs after infection of GFP Control Adenovirus (6x10 9 vp/ml) on 12 well plate (0.75x10 6 cells/well) in hepatocyte cells 10 Vigene Biosciences

11 Lentivirus overview and CAR-T Lentivirus is a subfamily of retroviruses. Lentiviruses can deliver a significant amount of RNA into the host cells including both dividing and non-dividing cells. In packaging lentivirus particles, 3 components are needed: virus RNA, internal structural and enzymatic proteins, and the envelope glycoprotein. Third generation packaging systems contain only gag, pol, and rev genes. It utilizes a chimeric 5 LTR to ensure transcription in the absence of Tat. Third generation packaging method is the safest. Lentivirus is considered one of the most efficient methods for gene delivery. Several features make lentivirus preferable. Unlike other retroviruses, lentiviruses have the ability to infect both dividing and non-dividing cells. Lentiviral vectors can integrate their genetic cargo directly into the chromosome of the target cell but do not transfer sequences that encode for proteins derived from the packaging virus. This is key to preventing an immune response to the cells containing the transferred gene. Finally, lentiviral vectors can be pseudotyped to infect specific cell types only or a broad range of targets. Vigene Biosciences offers ultra high titer lentivirus production service. With our large scale production, the titer is usually > viral particles/ml measured by qpcr or IU/ml in HEK293 cells. CAR-T and lentivirus Chimeric Antigen Receptor (CAR) can be expressed on the surface of the T-cells, providing them with a specific cancer targeting mechanism. CAR+ T-cells = CAR-T. In CAR-T the first step consists of harvesting the T-cells from the patient (autologous therapy) or from a healthy person (allogeneic therapy). Then, these cells can be transduced via a vrial vector to give them a new sequence of DNA which codes for the new receptor CAR. To deliver the CAR into the T-cells, the most efficient way is via lentivirus-based delivery. AAV, Adenovirus and Lentivirus-based Gene Delivery Handbook 11

12 Benefits Integrative transduction Good for making stable cell lines Transduce both both dividing and non-dividing cells T- cells Lentivirus expression data Benefits transduction Integrative Caveats Good making stablebycell lines integration for Genetic footprint lentiviral Transduce both dividing and non-dividing cells cellsproducts and services TViGene Genome wide lentiviral FL human cdna viral Ultra high titer and purity Caveats particles footprint Lentiviral cloning and packaging Genetic by lentiviral integration Vigene products and services Genome wide lentiviral FL human cdna viral particles Lentiviral cloning and packaging Ultra high titer and purity 1 ul before concentration 1 ul after concentration HEK293 transducedwith withsame same volume (1viral µl viral stock to 2culture ml culture medium) HEK293cells cellswere were transduced volume (1 µl stock to 2 ml medium) ofofconcentrated lentivirus and of virus before purification. Images were taken 48 concentrated lentivirus and of virus before purification. Images were taken 48 hours hours after after transduction. The purified and concentrated dramatically increased the transduction. The purified and concentrated lentiviruslentivirus dramatically increased the intensity GFP expression and number of number GFP positive cells.positive cells. intensity GFP expression and of GFP 12 Vigene Biosciences

13 AAV, adenovirus and lentivirus cgmp production cgmp section Vigene Biosciences has the expertise to assist you with the cgmp manufacture of AAV, adenovirus or lentivirus vectors for clinical use. Our top quality system combined with the industry leading production technologies opens up exciting opportunities for both academia and industry in the clinical use of AAV, lentivirus and adenovirus. Lot sizes of 10^15 10^16 viral particles (AAV and adenovirus); 10^11 10^12 viral particles (lentivirus) ISO 7 and ISO 8 cleanrooms. Document Control Master Documents Standard Operating Procedures Material Specifications Product Specifications Batch Production Records Validation Protocols/Reports Working Document Issuance Executable forms and Batch Production Records Documents/Record Archive Material Control Generate Material Specifications for all components used in manufacture of GMP material, which identifies: Material Approval Manufacturers and Suppliers Catalog numbers Grade Storage conditions Handling and disposal procedures Equipment and Environment Control Maintenance SOPs and Equipment History Files for all equipment used in manufacture or support of GMP product Validation; Calibration; Maintenance; Use Cleaning HVAC- ISO 7 and ISO 8 Cleanrooms Environmental Monitoring Viable Air Viable Surface Non-viable Particulates Personnel Cleaning Alternating disinfectants Product Control Product Specifications under GMPs Minimum Release Criteria Sterility- Negative for Mycoplasma Batch Production Records and support documentation for compliance to GMPs, Internal procedures and batch record instructions Certificates of Analysis with each GMP batch Cross Contamination Prevention Dedicated Production Areas AAV, Adenovirus and Lentivirus-based Gene Delivery Handbook 13

14 Tissue-specific promoters compatible with AAV, adenovirus and lentivirus Cat.# Promoter Size Description PM10001 ALB 2.4kb Liver specific; 10 timer stronger than CMV after 10 weeks PM10002 GFAP bp Hybrid of EF1a and GFAP PM10003 CAG 944bp Strong promoter, ubiquitous expression in vivo PM10004 CamKIIa 1.2kb Specific expression in excitatory neurons in the neocortex and hippocampus PM10005 EF1A 1.2kb Ubiquitously expressed; weaker than CMV but better for in vivo PM10006 CK kb PM10007 CK bp Calcium/Calmodulin-dependent kinase II alpha PM10008 GFAP 2.0kb Astrocyte-specific expression PM10009 MBP 1.3kb Myelin basic protein promoter; efficient transduction of oligodendrocytes by AAV type 8 vectors PM10010 EFFS 253bp A short version EF1A PM10011 TBG 460bp Homo sapiens serpin peptidase inhibitor, clade A PM10012 amhc 0.4kb Mouse myosin heavy chain alpha promoter PM10013 ctnt 702bp Specifically transduce cardiomyocytes PM10014 Synapsin 471bp Specific in neuron PM10015 Mecp2 230bp Truncated Mcep2 neuron specific PM10016 c-fos 1.7kb Activity-dependent promoter PM10017 MCK 1.35kb Muscle creatine kinase promoter/enhancer PM10018 UBC 1.1kb Ubiquitous, weaker than CMV but better for in vivo PM10019 PGK 400bp Ubiquitous, weaker than CMV but better for in vivo PM10020 Somatostat 1.2kb Expression restricted to GABAergic neuron PM10021 Rpe65 700bp Retinal Pigment epithelium-specific expression in vivo and in vitro PM10022 Insulin1 1.0kb Specifically expressed in beta- cells of the pancreas PM XEnhancer MCK 728bp Much stronger than CMV in muscle, inactive in nonmuscle cell lines and mouse liver PM10025 NSE 1.3kb Neuron-specific enolase promoter 14 Vigene Biosciences

15 Vigene AAV products, adenovirus product and service guide Delivery system Products /services Titer and volume or quantity Price AAV Ready-to-package AAV shrna for human genes Custom small scale AAV packaging projects Custom large scale AAV packaging projects 4 AAV shrna plasmids+ 1 scrambled control $ ul of >10^12 GC /ml $ ul of >10^13 GC /ml $1398 cgmp AAV vector production project 10^15-10^16 GC Inquire Adenovirus Premade adenovirus for full length human cdna expression 500ul of >10^10 GC /ml Starting from $498 Custom large scale adenovirus packaging projects 500ul of >10^12GC /ml $1398 cgmp adenovirus packaging project 10^13-10^15 GC Inquire Lentivirus Read-to-use lenti virus for full length human cdna and mirna expression 100ul of lentivirus > 10^9 GC/ml * Starting from $498 Custom small scale lentivirus packaging projects 100ul of lentivirus; > 10^9 GC/ml $595 Ultra high titer custom lentivirus packaging service 200ul of lentivirus >10^10-10^11 GC/ml $1495 cgmp lentivirus packaging project 10^11-10^12 GC Inquire * The titer of lentivirus is very sensitive to the size of viral genome. The titer offered here is based on the assumption that the insert between two LTRs is less than 5.1kb and the gene of interest is less than 1.5kb. For every kb-long increase of the viral genome, the titer will be decreased by a factor of 10. Genome copy (GC) is measured by qpcr of lentivirus. AAV, Adenovirus and Lentivirus-based Gene Delivery Handbook 15

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