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Dive into the research topics where Pavitra Roychoudhury is active.

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Featured researches published by Pavitra Roychoudhury.


PLOS ONE | 2014

AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication

Nicholas D. Weber; Daniel Stone; Ruth Hall Sedlak; Harshana S. De Silva Feelixge; Pavitra Roychoudhury; Joshua T. Schiffer; Martine Aubert; Keith R. Jerome

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.


Antiviral Research | 2016

Detection of treatment-resistant infectious HIV after genome-directed antiviral endonuclease therapy

Harshana S. De Silva Feelixge; Daniel Stone; Harlan L. Pietz; Pavitra Roychoudhury; Alex L. Greninger; Joshua T. Schiffer; Martine Aubert; Keith R. Jerome

Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One potential approach to cure persistent viral infections is via the use of targeted endonucleases. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of HIV pol-specific zinc finger nucleases (ZFNs) alone or in combination with three prime repair exonuclease 2 (Trex2), we identified a treatment-resistant and infectious mutant virus that was derived from a ZFN-mediated disruption of reverse transcriptase (RT). Although gene disruption of HIV protease, RT and integrase could inhibit viral replication, a chance single amino acid insertion within the thumb domain of RT produced a virus that could actively replicate. The endonuclease-resistant virus could replicate in primary CD4(+) T cells, but remained susceptible to treatment with antiretroviral RT inhibitors. When secondary ZFN-derived mutations were introduced into the mutant viruss RT or integrase domains, replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.


Scientific Reports | 2016

Digital detection of endonuclease mediated gene disruption in the HIV provirus

Ruth Hall Sedlak; Shu Liang; Nixon Niyonzima; Harshana S. De Silva Feelixge; Pavitra Roychoudhury; Alexander L. Greninger; Nicholas D. Weber; Sandrine Boissel; Andrew M. Scharenberg; Anqi Cheng; Amalia Magaret; Roger E. Bumgarner; Daniel Stone; Keith R. Jerome

Genome editing by designer nucleases is a rapidly evolving technology utilized in a highly diverse set of research fields. Among all fields, the T7 endonuclease mismatch cleavage assay, or Surveyor assay, is the most commonly used tool to assess genomic editing by designer nucleases. This assay, while relatively easy to perform, provides only a semi-quantitative measure of mutation efficiency that lacks sensitivity and accuracy. We demonstrate a simple droplet digital PCR assay that quickly quantitates a range of indel mutations with detection as low as 0.02% mutant in a wild type background and precision (≤6%CV) and accuracy superior to either mismatch cleavage assay or clonal sequencing when compared to next-generation sequencing. The precision and simplicity of this assay will facilitate comparison of gene editing approaches and their optimization, accelerating progress in this rapidly-moving field.


Journal of Antimicrobial Chemotherapy | 2016

Pharmacodynamics of anti-HIV gene therapy using viral vectors and targeted endonucleases

Pavitra Roychoudhury; Harshana S. De Silva Feelixge; Harlan L. Pietz; Daniel Stone; Keith R. Jerome; Joshua T. Schiffer

OBJECTIVES A promising curative approach for HIV is to use designer endonucleases that bind and cleave specific target sequences within latent genomes, resulting in mutations that render the virus replication incompetent. We developed a mathematical model to describe the expression and activity of endonucleases delivered to HIV-infected cells using engineered viral vectors in order to guide dose selection and predict therapeutic outcomes. METHODS We developed a mechanistic model that predicts the number of transgene copies expressed at a given dose in individual target cells from fluorescence of a reporter gene. We fitted the model to flow cytometry datasets to determine the optimal vector serotype, promoter and dose required to achieve maximum expression. RESULTS We showed that our model provides a more accurate measure of transduction efficiency compared with gating-based methods, which underestimate the percentage of cells expressing reporter genes. We identified that gene expression follows a sigmoid dose-response relationship and that the level of gene expression saturation depends on vector serotype and promoter. We also demonstrated that significant bottlenecks exist at the level of viral uptake and gene expression: only ∼1 in 220 added vectors enter a cell and, of these, depending on the dose and promoter used, between 1 in 15 and 1 in 1500 express transgene. CONCLUSIONS Our model provides a quantitative method of dose selection and optimization that can be readily applied to a wide range of other gene therapy applications. Reducing bottlenecks in delivery will be key to reducing the number of doses required for a functional cure.


Journal of Immunology | 2018

A Fixed Spatial Structure of CD8+ T Cells in Tissue during Chronic HSV-2 Infection

Joshua T. Schiffer; Dave A. Swan; Pavitra Roychoudhury; Jennifer M. Lund; Martin Prlic; Jia Zhu; Anna Wald; Lawrence Corey

Tissue-resident CD8+ T cells (Trm) can rapidly eliminate virally infected cells, but their heterogeneous spatial distribution may leave gaps in protection within tissues. Although Trm patrol prior sites of viral replication, murine studies suggest they do not redistribute to adjacent uninfected sites to provide wider protection. We perform mathematical modeling of HSV-2 shedding in Homo sapiens and predict that infection does not induce enough Trm in many genital tract regions to eliminate shedding; a strict spatial distribution pattern of mucosal CD8+ T cell density is maintained throughout chronic infection, and trafficking of Trm across wide genital tract areas is unlikely. These predictions are confirmed with spatial analysis of CD8+ T cell distribution in histopathologic specimens from human genital biopsies. Further simulations predict that the key mechanistic correlate of protection following therapeutic HSV-2 vaccination would be an increase in total Trm rather than spatial reassortment of these cells. The fixed spatial structure of Trm induced by HSV-2 is sufficient for rapid elimination of infected cells but only in a portion of genital tract microregions.


bioRxiv | 2017

Genomic and proteomic analysis of Human herpesvirus 6 reveals distinct clustering of acute versus inherited forms and reannotation of reference strain

Alexander L. Greninger; Giselle M. Knudsen; Pavitra Roychoudhury; Derek Hanson; Ruth Hall Sedlak; Hong Xie; Jon Guan; Thuy Thuong Thuong Nguyen; Vikas Peddu; Michael Boeckh; Meei-Li Huang; Linda S. Cook; Daniel P. Depledge; Danielle M. Zerr; David M. Koelle; Soren Gantt; Tetsushi Yoshikawa; Mary T. Caserta; Joshua A. Hill; Keith R. Jerome

Human herpesvirus-6A and -6B (HHV-6) are betaherpesviruses that reach >90% seroprevalence in the adult population. Unique among human herpesviruses, HHV-6 can integrate into the subtelomeric regions of human chromosomes; when this occurs in germ line cells it causes a condition called inherited chromosomally integrated HHV-6 (iciHHV-6). To date, only two complete genomes are available for HHV-6B. Using a custom capture panel for HHV-6B, we report near-complete genomes from 61 isolates of HHV-6B from active infections (20 from Japan, 35 from New York state, and 6 from Uganda), and 64 strains of iciHHV-6B (mostly from North America). We also report partial genome sequences from 10 strains of iciHHV-6A. Although the overall sequence diversity of HHV-6 is limited relative to other human herpesviruses, our sequencing identified geographical clustering of HHV-6B sequences from active infections, as well as evidence of recombination among HHV-6B strains. One strain of active HHV-6B was more divergent than any other HHV-6B previously sequenced. In contrast to the active infections, sequences from iciHHV-6 cases showed reduced sequence diversity. Strikingly, multiple iciHHV-6B sequences from unrelated individuals were found to be completely identical, consistent with a founder effect. However, several iciHHV-6B strains intermingled with strains from active pediatric infection, consistent with the hypothesis that intermittent de novo integration into host germline cells can occur during active infection Comparative genomic analysis of the newly sequenced strains revealed numerous instances where conflicting annotations between the two existing reference genomes could be resolved. Combining these findings with transcriptome sequencing and shotgun proteomics, we reannotated the HHV-6B genome and found multiple instances of novel splicing and genes that hitherto had gone unannotated. The results presented here constitute a significant genomic resource for future studies on the detection, diversity, and control of HHV-6. Author Summary HHV-6 is a ubiquitous large DNA virus that is the most common cause of febrile seizures and reactivates in allogeneic stem cell patients. It also has the unique ability among human herpesviruses to be integrated into the genome of every cell via integration in the germ line, a condition called inherited chromosomally integrated (ici)HHV-6, which affects approximately 1% of the population. To date, very little is known about the comparative genomics of HHV-6. We sequenced 61 isolates of HHV-6B from active infections, 64 strains of iciHHV-6B, and 10 strains of iciHHV-6A. We found geographic clustering of HHV-6B strains from active infections. In contrast, iciHHV-6B had reduced sequence diversity, with many identical sequences of iciHHV-6 found in individuals not known to share recent common ancestry, consistent with a founder effect from a remote common ancestor with iciHHV-6. We also combined our genomic analysis with transcriptome sequencing and shotgun proteomics to correct previous misannotations of the HHV-6 genome.


Scientific Reports | 2017

In vivo dynamics of AAV-mediated gene delivery to sensory neurons of the trigeminal ganglia

Chung H. Dang; Martine Aubert; Harshana S. De Silva Feelixge; Kurt Diem; Michelle Loprieno; Pavitra Roychoudhury; Daniel Stone; Keith R. Jerome

The ability to genetically manipulate trigeminal ganglion (TG) neurons would be useful in the study of the craniofacial nervous system and latent alphaherpesvirus infections. We investigated adeno-associated virus (AAV) vectors for gene delivery to the TG after intradermal whiskerpad delivery in mice. We demonstrated that AAV vectors of serotypes 1, 7, 8, and 9 trafficked from the whiskerpad into TG neurons and expressed transgenes within cell bodies and axons of sensory neurons in all three branches of the TG. Gene expression was highest with AAV1, and steadily increased over time up to day 28. Both constitutive and neuronal-specific promoters were able to drive transgene expression in TG neurons. Levels of vector genomes in the TG increased with input dose, and multiple transgenes could be co-delivered to TG neurons by separate AAV vectors. In conclusion, AAV1 vectors are suitable for gene delivery to TG sensory neurons following intradermal whiskerpad injection.


Protein Engineering Design & Selection | 2017

Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses.

Nixon Niyonzima; A.R. Lambert; R. Werther; H. De Silva Feelixge; Pavitra Roychoudhury; Alexander L. Greninger; Daniel Stone; Barry L. Stoddard; Keith R. Jerome

The combination of yeast surface display and flow cytometric analyses and selections is being used with increasing frequency to alter specificity of macromolecular recognition, including both protein-protein and protein-nucleic acid interactions. Here we describe the use of yeast surface display and cleavage-dependent flow cytometric assays to increase the specificity of an engineered meganuclease. The re-engineered meganuclease displays a significantly tightened specificity profile, while binding its cognate target site with a slightly lower, but still sub-nanomolar affinity. When incorporated into otherwise identical megaTAL protein scaffolds, these two nucleases display significantly different activity and toxicity profiles in cellulo. The structural basis for reprogrammed DNA cleavage specificity was further examined via high-resolution X-ray crystal structures of both enzymes. This analysis illustrated the altered protein-DNA contacts produced by mutagenesis and selection, that resulted both in altered readout of those based and a necessary reduction in DNA binding affinity that were necessary to improve specificity across the target site. The results of this study provide an illustrative example of the potential (and the challenges) associated with the use of surface display and flow cytometry for the retargeting and optimization of enzymes that act on nucleic acid substrates in a sequence-specific manner.


Molecular Therapy | 2016

765. Detection of Treatment-Resistant Infectious HIV After Genome-Directed Antiviral Endonuclease Therapy

Daniel Stone; Harshana S. De Silva Feelixge; Harlan L. Pietz; Pavitra Roychoudhury; Alex L. Greninger; Joshua T. Schiffer; Martine Aubert; Keith R. Jerome

Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One newly emerging approach to cure persistent viral infections is via the use of targeted endonucleases. To date, endonucleases have been used as therapeutic agents targeting the genomes of viruses including EBV, JCV, HBV, HCV, HIV, HPV, HSV and HTLV, and have been shown to inhibit viral replication and subsequently persistence. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of a panel of four HIV pol-specific zinc finger nucleases (ZFNs) we identified a provirus encoding a treatment-resistant and infectious mutant virus that was derived from a ZFN2-mediated disruption of reverse transcriptase (RT). Although ZFN-mediated disruption of HIV protease (ZFN1), RT (ZFN2 & ZFN3) and integrase (ZFN4) coding sequences could inhibit viral replication, a RT mutant provirus was detected that produced a replication competent and ZFN2 cleavage resistant HIV. Mutant virus ZFN2(+3) contained a single amino acid insertion that introduced a Leucine-Leucine (LL) motif in the thumb domain of RT. The RT of mutant virus ZFN2(+3) likely remained functional since a LL motif is also found in the RT of several hominid, old world monkey or equine spumaviruses. In ZFN2-treated HEK293 and CD4+ SupT1 T cells, we found that up to 25% and 6% of all ZFN2 HIV pol target site mutations detected by Illumina sequencing encoded the ZFN2(+3) mutant respectively. We found that ZFN2(+3) mutant virus could replicate at levels comparable to wild type HIV in primary CD4+ T cells, but importantly remained susceptible to treatment with both NRTi and NNRTi antiretroviral inhibitors. When secondary ZFN-derived mutations were introduced into other RT or integrase domains of mutant virus ZFN2(+3), replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.


JCI insight | 2016

In vivo disruption of latent HSV by designer endonuclease therapy

Martine Aubert; Emily A. Madden; Michelle Loprieno; Harshana S. DeSilva Feelixge; Laurence Stensland; Meei-Li Huang; Alexander L. Greninger; Pavitra Roychoudhury; Nixon Niyonzima; Thuy Nguyen; Amalia Magaret; Roman Galleto; Daniel Stone; Keith R. Jerome

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Keith R. Jerome

Fred Hutchinson Cancer Research Center

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Daniel Stone

Fred Hutchinson Cancer Research Center

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Harshana S. De Silva Feelixge

Fred Hutchinson Cancer Research Center

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Joshua T. Schiffer

Fred Hutchinson Cancer Research Center

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Martine Aubert

Fred Hutchinson Cancer Research Center

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Harlan L. Pietz

Fred Hutchinson Cancer Research Center

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Meei-Li Huang

Fred Hutchinson Cancer Research Center

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Nixon Niyonzima

Fred Hutchinson Cancer Research Center

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