Research Interests
The major goals of our laboratory are to comprehensively understand the biology of recombinant adeno-associated virus (AAV) and to develop new AAV vector-mediated gene and cell therapies to treat various human diseases. Scientific curiosity is the most important, major driver of the lab, having led to many discoveries and breakthroughs in AAV vectors and gene therapy. We also fully enjoy leveraging our knowledge and expertise around AAV, virus and gene therapy research to understand broader and more fundamental biological questions that have yet to be addressed or answered in various scientific disciplines.
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- AAV & AAV Biology
- Data-driven Research
- AAV Vector Development
- Gene Therapy
AAV & AAV Biology
AAV and AAV Biology
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AAV belongs to the parvovirus family and is the smallest virus that has the simplest structure. The virus is 25 nm in diameter and is composed of a protein shell, called capsid, and a 4.7-kb single-stranded DNA genome coding two viral genes, the rep and cap genes. The rep gene encodes viral non-structural proteins, Rep40, Rep52, Rep68 and Rep78, translated from a single open reading frame (ORF), and play roles in viral genome replication and packaging. The cap gene encodes structural proteins VP1, VP2 and VP3 translated from a single ORF and two non-structural proteins, AAP (assembly activating protein) and MAAP (membrane-associated accessory protein) from +1 frameshifted ORFs. VP proteins form the viral capsid made of 60 VP subunits at a ratio of VP1(1):VP2(1):VP3(10). The AAV genome has inverted terminal repeats (ITRs) at both genome termini that form a secondary hairpin structure as a result of their palindromic sequences. AAV infects humans but does not cause any human diseases; therefore, AAV is nonpathogenic and offers an attractive vehicle to deliver therapeutic genes to human body.
AAV infects cells via cell surface viral receptors, enter cells, pass the nuclear envelop and disassemble in the nucleus, releasing the viral genome and establishing latent infection in non-dividing cells. In replicating cells, AAV viral genomes do not replicate or minimally replicate and therefore are lost eventually. A small portion of the viral genomes integrate into the host cellular DNA. In AAV serotype 2 (AAV2), viral genomes integrate into the AAVS1 site on human chromosome 19 in a site-specific fashion by a Rep-mediated mechanism. However, the current view is that this is not likely the mechanism of AAV2 latent infection in the human body, and AAV viral genomes remain as episomes (extrachromosomal viral genomes) for a long period of time to maintain latency. Upon coinfection or superinfection with adenovirus or other helperviruses such as herpes simplex virus and cytomegalovirus, the AAV enters the productive viral life cycle, replicating viral genomes and producing progeny.
At least 13 AAV serotypes have been identified and hundreds of naturally occurring AAV variants have been isolated in various organs from humans and non-human primates. Each serotype and naturally occurring variant exhibits unique biological properties and show serotype/variant-dependent tropism during the infection process.
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Cloning of the AAV2 genome into a plasmid backbone provided a model for understanding of the AAV virus life cycle. The discovery that the inverted terminal repeats (ITRs) were the only DNA sequences required in cis for AAV production in cultured cells led to the use of recombinant AAV (AAV) as a vector for gene therapy. In recombinant AAV vectors that deliver to cells a gene of interest (GOI) including therapeutic genes, the rep and cap genes are removed from the AAV viral DNA genome and replaced with GOI, leaving AAV ITRs as the only viral genome-derived component in recombinant AAV vector genome. AAV vectors can be produced in packaging cells (e.g., human embryonic kidney (HEK) 293 cells) by delivering AAV vector plasmid, AAV helper plasmid and adenovirus (Ad) helper plasmid to the cells by transient DNA transfection. The AAV vector plasmid provides an AAV vector genome to be packaged in the virion, the AAV helper plasmid supplies AAV Rep, VP and AAP proteins, and the Ad helper plasmid supplies adenovirus components essential for virus replication and production in cells.
Specific areas of interest
An understanding of basic AAV biology has been accomplished over five decades of studies, yet a number of complex questions remain unanswered, including how AAV capsids are formed, how AAV viral genomes are packaged into the capsid shell, how viral proteins and host proteins interact in the process of virion formation, how AAV enters the cells via receptor binding and endocytosis, traffics to the nucleus and uncoats releasing viral genome, how AAV viral genomes are processed via the host cell DNA damage responses and repair mechanisms, how viral capsid proteins, viral genome and host transcription/translation machinery interact enhancing and suppressing transgene expression, how capsid amino acid sequence and structure determine AAV biological properties, among others.
How did the overlapping AAP and VP ORFs co-evolve?
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A proposed model of AAP2 ORF overprinting in the AAV2VP ORF deduced by evolutionary computation. The evolutionarily conserved QVKEVTQ and KSKRSRR motifs, a pair of overlapping heptapeptides in VP and AAP of AAV2, respectively, were randomized and experimentally evolved under no coevolutionary constraints, and a large number of capsid-forming VP mutants and functionally competent AAP mutants of these motifs were identified by the next-gen sequencing. The obtained heptapeptide information was then integrated into an evolutionary algorithm, with which VP and AAP were computationally co-evolved from random nucleotide sequences in silico. This experimental and computational evolution-based analysis revealed how overlap-evoked co-evolutionary constraints play a role in making the VP and AAP heptapeptide sequences into the present shape and led to a hypothetical model of VP and AAP co-evolution as depicted in this figure. The most primitive heptapeptide sequence in the AAV2 VP protein when no AAP existed is shown at the top left. The heptapeptide region in the present shape is shown at the right bottom.
Kawano, Y., Neeley, S., 1, Adachi, K., Nakai, H. (2013) An experimental and computational evolution-based method to study a mode of co-evolution of overlapping open reading frames in the AAV2 viral genome. PLoS One 8, e66211.
How does AAP contribute to capsid assembly?
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Heterogeneity of the roles of AAV in the process of AAV capsid assembly among different AAV serotypes. (Left Panel) Subcellular localization of AAP and assembled capsids in HEK293 cells. AAV2 and AAV4 assemble in the nucleolus while AAV5, AAV8 and AAV9 assemble in the nucleoplasm outside the nucleolus. (Right Panel) AAV VP3 only capsid production with or without AAP. AAV2 capsid formation requires AAP while AAV5 capsid formation does not.
Earley, L.F., Powers, J.M, Adachi, K., Baumgart, J., Meyer, N.L., Xie, Q., Chapman, M.S., Nakai, H. (2017) Adeno-associated virus assembly-activating protein is not an essential requirement for capsid assembly of AAV serotypes 4, 5 and 11. J. Virol. 91:e01980-16
How is the genome packaging capacity determined?
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Methods of calculation of AAV capsid inner volume and determination of genome packaging capacity of the AAV capsid. (Top Panels) A computational algorithm has been developed to precisely calculate capsid inner volume. For this purpose, pseudoatoms and inflated atoms are used to flatten irregular outer surface of the capsid and fill interstices present inside protein molecules, respectively.
What cellular proteins interact with AAV proteins in the process of capsid assembly?
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BioID approach to identify AAP-interacting cellular proteins (Top Panel). BioID proximity labeling followed by LC-MS/MS identifies AAP-interacting cellular proteins (e.g., deubiquitinating enzyme (DUB)). A preliminary hypothetical model of the interaction between DUB and AAV proteins in the process of capsid assembly (Bottom Panel).
How are the multifaceted biological phenotypes of AAV vectors governed in a system?
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Multi-faceted biological functions of the AAV capsid protein. AAV capsid VP protein has multifaceted functions in the tertiary and quaternary structures and contains multiple functional modules and components that interact with each other in a system, leading to the manifestation of emergent properties. Note: this is how viruses can make the most of its limited structural components to maximize its functions. It is very challenging to understand the system that governs virus phenotypes by traditional approaches. We have been addressing this question by using data-driven systems biology approaches (see “Data-driven Research” tab).