Establishment of adeno-associated virus latency by the recruitment of KAP1
Lead Research Organisation:
King's College London
Department Name: Immunology Infection and Inflam Diseases
Abstract
Viruses have evolved mechanisms that allow them to control and take advantage of the host cells, which they infect. We are studying a parvovirus, named adeno-associated virus (AAV) that adds an additional layer of complexity by exploiting and controlling a helper virus, which it needs to be able to replicate in cells. We have discovered that in the absence of helper viruses AAV associates with a cellular protein to silence its genome and that the virus can undo this by mediating a modification of the cellular protein that abolishes its silencing activity. Here we propose experiments that will allow us to determine the mechanism by which the cellular protein, KAP1, silences the viral genome. We will also investigate if helper viruses play a role in releasing KAP1 from the AAV genome and thereby allow production of AAV proteins. These products in turn will further modify KAP1 in order to avoid that AAV is silenced again. We will perform experiments to discern how AAV introduces changes to KAP1, which lead to its inactivation. The studies we propose will lead to a better understanding of the AAV life cycle and will provide a framework to study pathogenic parvoviruses such as B19, which can cause severe disease in patients with inherited blood disorders such as sickle cell anemia. Improved knowledge of silencing and reactivation of parvoviruses and by extension possibly other DNA viruses may lead to the development of therapies that control infections. In addition, our findings may also inform how production of gene therapy vectors derived from AAV can be improved. Recent clinical trials have demonstrated safety and efficacy of AAV-mediated gene therapy, however more efficient vector production schemes will be required to be able to offer gene therapy to large patient populations.
Technical Summary
Adeno-associated virus (AAV) is a human dependovirus that appears to have evolved an optimal working relationship with host and helper virus. With the support of viruses such as adenovirus and herpes virus, AAV replicates successfully, causing a cytopathic effect only in those cells which were already destined to suffer from helper virus infection; in the absence of co-infection the virus remains latent without causing any apparent harm. It was generally thought that latency is established through integration into the human genome, however our preliminary data indicate that latency of AAV might already be established prior to integration through interactions with the transcriptional co-repressor KAP1, which render the virus 'invisible' until conditions for replication are met. In addition, we have found that relief of repression is achieved in part through actions of the AAV Rep proteins, which lead to phosphorylation of KAP1. We propose experiments to further investigate the mechanism of KAP1-mediated repression of AAV, to determine a role for helper factors in the release of KAP1-mediated repression of the AAV genome and unravel the mechanism of Rep-mediated phosphorylation of KAP1. Knowledge gained from this proposal does not only establish a new concept in AAV biology but will also inform virus-host interactions of other DNA viruses, including pathogenic viruses. In addition, AAV has become a frontrunner as a vector for human gene therapy based on results from a number of clinical trials. However, the research community around AAV biology has remained very small and thus limited molecular insights into its life cycle is available. As an example of how an understanding of the biology of AAV can further the application of AAV vectors is demonstrated by our finding that KAP1 knockdown leads to increased recombinant AAV vector replication, an observation that could be exploited to help overcome the challenges of production for wide clinical use.
Planned Impact
This proposal focuses on the study of the interactions between the viral Rep protein and KAP1, a cellular protein that functions as a transcriptional co-repressor by recruiting histone modifying enzymes and heterochromatin-inducing factors. We have discovered that KAP1 associates with the AAV genome and helps establish viral latency. We propose to investigate how latency is established and how the virus has evolved mechanisms to escape from KAP1-mediated repression. Our work introduces a paradigm shift in the view of the life cycle of AAV and potentially other - pathogenic- parvoviruses such as B19. The molecular mechanisms by which B19 produces latency and reactivation are currently unknown. Given the strong similarities between the AAV Rep and the B19 NS1 protein, it is highly likely that KAP1 also plays a role in the life cycle of this pathogenic virus. We could envisage that by manipulating these interactions, treatments that curtail B19 replication in patients with inherited hemolytic anemia could be developed.
The proposed research also opens up possibilities for biotechnological developments that lead to the improvement of AAV gene therapy vector production schemes. We are currently investigating if manipulation of this pathway, potentially in combination with others, leads to a significant increase in recombinant AAV production. We already have established a collaboration with a major pharmaceutical company, and inclusion of a biotechnological translation of this project could be further explored with them.
We are communicating our research results through public access journals, teaching of pre-university and undergraduate students, lay audiences and through interactions with industrial partners.
The beneficiaries of this research potentially include: 1) pharmaceutical industry/biotech companies who have an interest in applying our research for the development of new therapies, 2) patients with diseases that could be treated with gene therapy/ patients with inherited hemolytic anemia such as sickle cell disease, 3) pre-university and university students who have an interest in pursuing a career in science, 4) the postdoctoral associate/Ph.D. student who will receive further training and profit from interactions with industrial partners.
The proposed research also opens up possibilities for biotechnological developments that lead to the improvement of AAV gene therapy vector production schemes. We are currently investigating if manipulation of this pathway, potentially in combination with others, leads to a significant increase in recombinant AAV production. We already have established a collaboration with a major pharmaceutical company, and inclusion of a biotechnological translation of this project could be further explored with them.
We are communicating our research results through public access journals, teaching of pre-university and undergraduate students, lay audiences and through interactions with industrial partners.
The beneficiaries of this research potentially include: 1) pharmaceutical industry/biotech companies who have an interest in applying our research for the development of new therapies, 2) patients with diseases that could be treated with gene therapy/ patients with inherited hemolytic anemia such as sickle cell disease, 3) pre-university and university students who have an interest in pursuing a career in science, 4) the postdoctoral associate/Ph.D. student who will receive further training and profit from interactions with industrial partners.
People |
ORCID iD |
Els Henckaerts (Principal Investigator) |
Publications
Raouf R
(2018)
Inhibition of somatosensory mechanotransduction by annexin A6.
in Science signaling
Meier A
(2021)
Herpes Simplex Virus 1 Coinfection Modifies Adeno-associated Virus Genome End Recombination
in Journal of Virology
Meier, Anita Felicitas
(2021)
Herpes Simplex Virus 1 Coinfection Modifies Adeno-associated Virus Genome End Recombination
Karda R
(2020)
Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors.
in Scientific reports
Smith-Moore S
(2018)
Adeno-associated virus Rep proteins antagonize phosphatase PP1 to counteract KAP1 repression of the latent viral genome.
in Proceedings of the National Academy of Sciences of the United States of America
Tordo J
(2018)
A novel adeno-associated virus capsid with enhanced neurotropism corrects a lysosomal transmembrane enzyme deficiency.
in Brain : a journal of neurology
Description | Pfizer Rare Disease Consortium Award |
Amount | £1,049,600 (GBP) |
Organisation | Pfizer Inc |
Sector | Private |
Country | United States |
Start | 09/2015 |
End | 09/2018 |
Title | BioID AAV Rep |
Description | List of cellular proteins that interact with the various AAV Rep proteins as discovered via a BioID approach |
Type Of Material | Database/Collection of data |
Year Produced | 2016 |
Provided To Others? | Yes |
Impact | This database may lead to the discovery of new virus-host interactions. In addition, it may lead to the discovery of cellular factors that could be manipulated to increase recombinant AAV production. |
Description | Collaboration with Dr. Helen Rowe |
Organisation | University College London |
Department | Division of Infection and Immunity |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Expertise |
Collaborator Contribution | Reagents, protocols and expertise |
Impact | Publication: PNAS, in press |
Start Year | 2016 |
Description | Collaboration with Prof. Cornel Fraefel |
Organisation | University of Zurich |
Country | Switzerland |
Sector | Academic/University |
PI Contribution | Reagents, expertise and protocols |
Collaborator Contribution | Reagents, expertise and protocol. In addition, one of Prof. Fraefel's PhD students is currently working in my lab; she is funded by a Doc Mobility grant from the Swiss National Science Foundation (duration: 1 year). |
Impact | Publication (PNAS, in press) and Doc Mobility fellowship awarded to Anita Meier |
Start Year | 2016 |
Description | Collaboration with Prof. Mathieu Bollen KU Leuven, Belgium |
Organisation | University of Leuven |
Country | Belgium |
Sector | Academic/University |
PI Contribution | Reagents, intelectual input and publication (PNAS, in press) |
Collaborator Contribution | Reagents, protocols and intelectual input |
Impact | Publication (PNAS in press) |
Start Year | 2017 |
Description | Society for Medicines Research, Meeting, Gene Therapy: Right Here, Right Now |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | My presentation focussed on the importance of understanding the biology of wild type adeno-associated virus (AAV) to further improve AAV-mediated gene therapy. The talk sparked an intense discussion; several members of the audience (pharmaceutical industry, regulators and academics) approached me to thank me for bringing up an aspect that has not been sufficiently addressed. The topic was further discussed during coffee/lunch sessions. |
Year(s) Of Engagement Activity | 2017 |
URL | https://www.smr.org.uk/smr/default.asp |
Description | Vist to Henrietta Barnet school |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | I gave a presentation about my career choices/career progress and presented an overview of my research; this was organised by the Biology Society. Several students approached me with questions about medical research career versus medical career and the prospects of becoming physician scientists. |
Year(s) Of Engagement Activity | 2017 |
Description | Work placement |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | 2- week work placement for year 10 student |
Year(s) Of Engagement Activity | 2017 |
Description | Work placement |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | 2-week work placement for year 10 student |
Year(s) Of Engagement Activity | 2016 |