Choosing Between Lentivirus and Adeno-associated Virus For DNA Delivery
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1 Choosing Between Lentivirus and Adeno-associated Virus For DNA Delivery Presenter: April 12, 2017 Ed Davis, Ph.D. Senior Application Scientist GeneCopoeia, Inc.
2 Outline Introduction to GeneCopoeia Lentiviral technology GeneCopoeia Lentiviral products & services AAV technology GeneCopoeia AAV products & services Which should I choose?
3 Outline Introduction to GeneCopoeia Lentiviral technology GeneCopoeia Lentiviral products & services AAV technology GeneCopoeia AAV products & services Which should I choose?
4 GeneCopoeia products & services Functional Genomics & Cell Biology Clones Viral systems Kits & reagents Fluorescent detection ORF Promoter mirna CRISPR shrna Lentivirus AAV Transfection Luciferase FISH probes Indel detection Cloning Cell function assays Nucleic acid detection Cell structure probes Fluorescent dyes
5 Outline Introduction to GeneCopoeia Lentiviral technology GeneCopoeia Lentiviral products & services AAV technology GeneCopoeia AAV products & services Which should I choose?
6 Why use viruses for DNA delivery? DNA transfection not always possible or practical. Some cell lines difficult or impossible to transfect. Necessary for in vivo/gene therapy applications Most cells support viral infection
7 Applications for viral DNA delivery Protein expression via cdna or open reading frame (ORF) clones Gene knockdown via RNA interference (RNAi) Reporter assays (GFP, luciferase) Gene editing via clustered, regularly-interspaced, short palindromic repeats- Cas9 (CRISPR-Cas9) Have been successfully carried out in cultured immortalized mammalian cell lines, primary cell culture, animal models, and in gene therapy on human patients
8 Lentivirus technology Class of retroviruses that includes human immunodeficiency virus (HIV) Single stranded RNA genome of 9.7 kb Integrates into genomic DNA Samulski & Muzyczka (2014). Annu. Rev. Virol. 1, 427. Infect dividing & nondividing cells
9 Lentivirus technology 3 rd generation lentivirus Sakuma, et al. (2012). Biochem. J. 443, 603.
10 Outline Introduction to GeneCopoeia Lentiviral technology GeneCopoeia Lentiviral products & services AAV technology GeneCopoeia AAV products & services Which should I choose?
11 Lentivirus technology GeneCopoeia lentiviral plasmids
12 Lentivirus technology GeneCopoeia lentiviral plasmids
13 GeneCopoeia Lentiviral products and services Product/service Lentiviral clones and cloning vectors Lentifect lentiviral particles Lenti-Pac Lentiviral Packaging Reagents Description Pre-made and custom clones carrying ORFs, promoters, shrnas, mirna 3 UTRs, precursors, and inhibitors, sgrnas, and more. Available with multiple promoters, tags and reporters. Vectors for do-it-yourself cloning of sequences of interest. Pre-made and custom-packaged, ready to use lentiviral particles. Produced from GeneCopoeia s extensive, genome-wide clone collections or from customer-submitted clones. Complete system of reagents for do-it-yourself lentiviral particle production. Includes packaging plasmids, packaging cell line, particle concentration solution, and titration kit.
14 GeneCopoeia Lentiviral products and services Features Infect nearly all mammalian cell types Can be used to deliver relatively large DNA sequences-up to about 5-6 kb in length Can be used to generate stable cell lines, or drive stable gene expression in organs and tissues in vivo, due to integration of the transgene at random locations in the genome
15 Outline Introduction to GeneCopoeia Lentiviral technology GeneCopoeia Lentiviral products & services AAV technology GeneCopoeia AAV products & services Which should I choose?
16 AAV technology Identified as contaminant of adenovirus (Ad) Single stranded DNA genome of 4.7 kb Requires helper virus for infection Integrates into genomic DNA Infects dividing & nondividing cells Ran, et al. (2013). Nature Protocols 8, 2281
17 AAV technology AAV modifications Transgene plasmid ITR Gene ITR Cap & Rev in trans Cap Rep Ad helper genes VA E4 E2A Moved Cap & Rep genes to different plasmid-can accommodate inserts up to 4 kb (<3 kb is ideal)
18 AAV technology Serotypes Determined by proteins in capsid Influences tissue infectivity. 13 serotypes currently in use. Most common serotype is AAV-2, due to broad tissue infectivity Provides ability to restrict infection to specific tissues or cell types, if desired
19 AAV technology: Serotypes Serotype Primary target tissue Description AAV-1 Muscle Best for cardiac muscle, skeletal muscle, neuronal and glial tissue. AAV-2 Muscle, Liver, Retina Most commonly-used serotype. Best for neurons, muscle, liver, and brain. AAV-3 Megakaryocytes Best for megakaryocytes, muscle, liver, lung, and retina. AAV-4 Retina Best for neurons, muscle, brain, and retina. AAV-5 Lung Best for lung, neurons, synovial joint, retina, and pancreas. AAV-6 Muscle, Lung Best for lung, liver, and heart. AAV-7 Muscle, Retina, Neurons Best for muscle, neurons, and liver. AAV-8 Liver Best for muscle, brain, liver, and retina. AAV-9 Various Best for muscle, heart, liver, lung, and brain. AAV-10 AAV-DJ Pleura, CNS Various Cloned from Cynomolgus, almost identical with AAVrh10 except for 12 amino acids in VP1. Best for lung, muscle, heart, NCS and liver. A mixture of 8 naturally-occurring serotypes. Efficiently transduces a wide variety of cell types in vitro. AAV-DJ/8 Various A variant of AAV-DJ with a heparin binding domain (HBD) mutation, which permits infection of liver as well as other tissues in vivo.
20 Outline Introduction to GeneCopoeia Lentiviral technology GeneCopoeia Lentiviral products & services AAV technology GeneCopoeia AAV products & services Which should I choose?
21 GeneCopoeia AAV products and services Vector types
22 GeneCopoeia AAV products and services Packaging service Available in multiple vector types (CMV, CAG, or EF1a promoter), or you can submit your own vector Available for multiple serotypes. Available in standard purity (for in vitro use only; 1x10^11 GC/ml) or purified (for in vivo use; 5x10^12 GC/ml) Insert sizes must be < 3 kb
23 GeneCopoeia AAV products and services Features High titers. Titer of purified particles can be up to 10^14 GC/ml (genome copies/ml) Versatile. Usable in a broad range of host cell types Low toxicity. Does not integrate into the host genome. Low immunogenicity. Minimal host immune response. Safe. Not associated with any human disease
24 GeneCopoeia AAV products and services Performance of standard & purified particles A egfp RFP mcherry B μl 0.05 μl 0.5 μl
25 Outline Introduction to GeneCopoeia Lentiviral technology GeneCopoeia Lentiviral products & services AAV technology GeneCopoeia AAV products & services Which should I choose?
26 Which should I choose?
27 GeneCopoeia AAV products and services GeneCopoeia Technical Note: Lentivirus or AAV? Download from:
28 Upcoming webinar! Applications For Safe Harbor Transgenesis in Genome Editing Wednesday, April 19, :00 pm ET Register here:
29 Summary Lentivirus & AAV are invaluable for introducing genetic material into mammalian cells, either in culture, in whole animals, or in gene therapy Each viral system has its own unique advantages and disadvantages, depending on the application Lentivirus is most useful for its broad tissue tropism and ability to carry larger inserts AAV is most useful for its lack of toxicity and immunogenicity, and its natural ability to limit tissue type infection GeneCopoeia offers extensive products and services for lentivirus and AAV, including custom viral particle production.
30 Thank you! If you have any additional questions, please call x227 Or visit us on the web: GeneCopoeia, Inc Medical Center Drive Suite 101 Rockville, Maryland USA 20850
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