Adeno-Associated Viral (AAV) Vectors. Biosafety office

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1 Adeno-Associated Viral (AAV) Vectors Biosafety office April 2014

2 Copyright 2014 University of Cincinnati, All Rights Reserved. All rights reserved. No part of this publication may be reproduced, distributed, or transmitted, except for use in research and education, in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the University of Cincinnati. For permission requests, write to the University of Cincinnati, addressed Attention: Biosafety Officer, at the address below. THE UNIVERSITY OF CINCINNATI MAKES NO REPRESENTATIONS AND EXTENDS NO WARRANTIES OF ANY KIND, EITHER EXPRESS OR IMPLIED. THERE ARE NO EXPRESS OR IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, OR THAT THE PUBLICATION PROVIDED HEREUNDER SHALL BE FREE OF INFRINGEMENT OF THIRD-PARTY INTELLECTUAL PROPERTY RIGHTS OR OTHER RIGHTS. Biosafety Office University of Cincinnati 51 Goodman Dr. Cincinnati, OH inbiocom@ucmail.uc.edu

3 Adeno-Associated Virus AAV belongs to the Parvoviridae family and to the Dependovirus genus. It is a small non-enveloped icosahedral virus (20-25 nm in diameter). It is naturally a replication-defective virus. For replication, AVV dependents on the presence of wild type helper viruses, including Adenovirus (from which the AAV name originated), Herpes Simplex virus, Human Papilloma virus, Vaccinia virus or Cytomegalovirus. Virus Genome : The AAV genome consists of a single-stranded DNA (approximately 4.7 kb) which comprises Inverted Terminal Repeats (ITRs) at both ends, and two open reading frames (ORFs): rep and cap. The ITRs flank the two viral genes: rep (replication) and cap (capsid), encoding non-structural and structural proteins, respectively. Rep is composed of four overlapping genes encoding proteins required for the AAV life cycle (Rep 78,Rep 68, Rep 52 and Rep 40). Cap contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry. The ITRs (145bp each) serve as origin of replication and play a key role in viral genome integration into the host genome as well as in the subsequent rescue of viral.

4 AAV Cycle AAV undergoes productive infection when co-infected with a helper virus, such as adenovirus. This is characterized by genome replication, viral gene expression and virion production. When AAV infects a human cell in the absence of a helper virus, its gene expression program is autorepressed and latency is ensued by preferential integration of the virus genome into a region of the human chromosome 19 (AAVS1). This site-specific integration involves the AAV ITRs and Rep proteins (Rep78, Rep68). Chromosome 19 integration requires AAV Rep protein expression. When a latently infected cell is further infected with a helper virus, such as adenovirus, the AAV gene expression program is activated leading to the AAV Rep-mediated rescue (i.e.excision) of the provirus DNA from the host cell chromosome, followed by replication and packaging of the viral genome. AAV Transmission and Clinical Manifestations AAV may be transmitted through direct contact with an infected individual or through indirect contact with the contaminated environment. Transmission of AAV can occur through ingestion, inhalation of aerosolized droplets, mucous membrane contact and accidental injection (for example, as the result of a needle stick). AAV has not been shown to cause disease in humans even though the majority of the population has been exposed. Many adults have antibodies reactive against one or more AAV serotypes, a finding which is entirely consistent with early and repeated exposures to AAV and adenoviruses throughout life

5 Cell Tropism - AAV AAV can infect a wide range of dividing and non-dividing cells from a variety of mammalian cells. Tissue specificity is determined by the capsid serotype. AAV2 is the most widely used AAV serotype in viral vector production. AAV2 attachment is primary mediated by heparan sulphate proteoglycans (HSPG), while internalization is aided by co-receptors, such as αvβ5 integrin and fibroblast growth factor receptor 1 (FGFR-1). AAV2 presents natural tropism towards skeletal muscles, neurons, vascular smooth muscle cells and hepatocytes. It should be noticed that many other serotypes of AAV have been isolated. Among them, AAV1, AAV2, AAV5, as well as AAV7, 8 and 9 have been used for gene delivery studies. Altering Cell Tropism: Pseudotyping An important area in the development of AAV as a vector concerns the engineering of altered cell tropisms to narrow or broaden raav2-mediated gene delivery and to increase infection efficiency. Between various AAV serotypes, the difference in transducing efficiencies could be caused by difference of their respective receptor content on target cells. A strategy to alter raav tropism exploits the natural capsid diversity of other serotypes, by packaging recombinant AAV2 genomes into capsids derived from other AAV isolates. The commonly used approach employs hybrid transcomplementing constructs that encode rep from AAV2 whereas cap is derived from the other serotype displaying the cell tropism of choice.

6 AAV Vector Production AAV2 is the best characterized serotype and the serotype for which most gene transfer studies have been based upon. Typically, AAV vector particles are generated by transfecting packaging cells with a plasmid (AAV cis-plasmid) containing a cloned recombinant AAV genome composed of the transgene flanked by the AAV ITRs (1), and a separate construct expressing in trans the viral rep and cap genes (2). The adenovirus genes, such as E1A, E1B, E2A, E4ORF6 and VA RNA*, must be provided by either adenovirus infection or plasmid (3). Given that HEK293 cells, commonly used vector production cells, already contain the E1A/E1b gene, the helper genes that need to be provided by a plasmid are E2A, E4ORF6 and VA RNA. The final viral vector particle will not contain any of the genes provided by the helper plasmids, but still remains infective! *The VA (viral associated) RNA is a type of noncoding RNA found in adenovirus. It plays a role in regulating translation Using adenovirus in vector production Although the method of using wild-type adenovirus can be easily scaled up in cultures and produce AAV vectors with very high titers, it is very challenging to completely get rid of the adenovirus from AAV product, and contamination of wild-type adenovirus is highly undesirable in view of vector safety and specificity.

7 AAV Vector & Genome Integration In humans, the wild type virus inserts preferentially at a specific site in a region on the long arm of chromosome 19 (19q13-qter), termed the AAVS1 site. Because AAV vectors are devoid of Rep coding sequences, the property of site-specific integration is not retained. Instead, persistent expression of vector sequences may occur from extrachromosomal (episomal) sequences and, in lower frequency, from randomly integrated sequences. Some studies have shown that the integration of AAV-2 vector genomes is not completely random and occurs mainly into active genes. Insertional mutagenesis is still a concern with AAV vectors. Credit: krishnacreations / Fotolia AAV Vector Application Advantages Wild type virus is not associated to any disease Infect dividing and non-dividing cells Disadvantages Oncogenesis potential Small transgene capacity Efficient gene transfer Unlike adenoviruses, AAVs are well tolerated and do not cause a strong innate end response or cytotoxic T cell response Stable integration into the host genome with stable expression of the transgene

8 AAV Vector: Environmental Stability AAV particles are stable in a wide ph range (3 to 9) and can resist heating at 56 o C for 1 hour. Due to the high stability of the capsid, AAV can remain infectious for at least a month at room temperature following simple dessication or lyophilization. AAV, as well as other non-enveloped viruses, is quite resistant to alcohol disinfectants. A broader action disinfection method, such as10% fresh household bleach solution, must be considered. This may be followed by an alcohol wipe to lessen the corrosive nature of the germicide. AAV Vector: Laboratory Activities Containment Although AAV belongs to the Risk Group classification 1 (RG1), the appropriate biosafety level will primarily depend on the nature of the gene carried by the vector. Recombinant AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule can, in most cases, be handled at biosafety level 1 (BSL1). BSL2 lab If a helper virus is used during the production of the vector, BSL2/ABSL2 containment is required. The nature of the transgene is critical for containment determination

9 AAV Vector: Laboratory Activities Exposure Risks Transmission of AAV can occur through inhalation of aerosolized droplets, mucous membrane contact, ingestion and accidental injection. Respiratory Exposure - AAV vector manipulations should be conducted in a containment equipment such as a biological safety cabinet (BSC). When handling AAV-containing cultures outside of containment equipment, a respirator (e.g. N95 mask) should be worn. To determine proper fit, wearers must be fit tested to make sure they have selected the appropriate model and size. OSHA requires that every employee who wears a respirator receive an initial fit-test prior to using that respirator followed by annual tests. Use aerosol containment devices when centrifuging. These include sealed canisters that fit in the centrifuge bucket, covers for the centrifuge bucket, heat sealed tubes, or sealed centrifuge rotors. Rotors should be removed and opened inside a BSC. Mucosal Exposure - Laboratory personnel may also be exposed by direct contact of vector suspension (e.g. splash) with oral, ocular or nasal mucosa. Extreme care must be taken to avoid spilling and/or splashing infected materials, especially if working with high volumes (>10L). The use of face shields provides protection against ocular, nasal and oral mucosa exposure. Also, a combination of goggles and a respirator provides adequate protection (mucosal and respiratory).

10 Resources D. R Deyle and D. W Russell1,2- Adeno-associated virus vector integration - Curr Opin Mol Ther. Aug 2009; 11(4): Shyam Daya and Kenneth I. Berns - Gene Therapy Using Adeno-Associated Virus Vectors Clinical Microbiology Reviews, Oct. 2008, p D. M. McCarty, S.M. Young Jr. and R. J.Samulski - Integration of Adeno-Associated Virus(AAV) and Recombinant AAV Vectors - Annu. Rev. Genet : RH Smith - Adeno-associated virus integration: virus versus Vector Gene Therapy (2008) 15, M. S. Lambert - A Reassessment of Adeno-Associated Virus Vector Risks That Takes New Information on Insertional Mutagenesis into Account - Applied Biosafety Vol. 13, No. 1, 2008