Khamaikawin, Wannisa

Preferred name

Main Affiliation

Faculty of Medicine

wannisa.kh@kmitl.ac.th

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The clonal repopulation of HSPC gene modified with anti-HIV-1 RNAi is not affected by preexisting HIV-1 infection
(2020-07-01)
Suryawanshi, Gajendra W.
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Khamaikawin, Wannisa
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Wen, Jing
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Shimizu, Saki
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Arokium, Hubert
Despite advances in hematopoietic stem/progenitor cell (HSPC) transplant for HIV-1-infected patients, the impact of a preexisting HIV-1 infection on the engraftment and clonal repopulation of HSPCs remains poorly understood. We have developed a long terminal repeat indexing-mediated integration site sequencing (LTRi-Seq) method that provides a multiplexed clonal quantitation of both anti-HIV-1 RNAi (RNA interference) gene-modified and control vector-modified cell populations, together with HIV-1-infected cells-all within the same animal. In our HIV-1-preinfected humanized mice, both therapeutic and control HSPCs repopulated efficiently without abnormalities. Although the HIV-1-mediated selection of anti-HIV-1 RNAi-modified clones was evident in HIV-1-infected mice, the organ-to-organ and intra-organ clonal distributions in infected mice were indistinguishable from those in uninfected mice. HIV-1-infected cells showed clonal patterns distinct from those of HSPCs. Our data demonstrate that, despite the substantial impact of HIV-1 infection on CD4+ T cells, HSPC repopulation remains polyclonal, thus supporting the use of HSPC transplant for anti-HIV treatment.
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In vivo selection of anti-HIV-1 gene-modified human hematopoietic stem/progenitor cells to enhance engraftment and HIV-1 inhibition
(2024-02-07)
Guo, Qi
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Zhang, Jian
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Parikh, Keval
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Brinkley, Alexander
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Lin, Samantha
Hematopoietic stem/progenitor cell (HSPC)–based anti-HIV-1 gene therapy holds great promise to eradicate HIV-1 or to provide long-term remission through a continuous supply of anti-HIV-1 gene-modified cells without ongoing antiretroviral therapy. However, achieving sufficient engraftment levels of anti-HIV gene-modified HSPC to provide therapeutic efficacy has been a major limitation. Here, we report an in vivo selection strategy for anti-HIV-1 gene-modified HSPC by introducing 6-thioguanine (6TG) chemoresistance through knocking down hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression using RNA interference (RNAi). We developed a lentiviral vector capable of co-expressing short hairpin RNA (shRNA) against HPRT alongside two anti-HIV-1 genes: shRNA targeting HIV-1 co-receptor CCR5 and a membrane-anchored HIV-1 fusion inhibitor, C46, for efficient in vivo selection of anti-HIV-1 gene-modified human HSPC. 6TG-mediated preconditioning and in vivo selection significantly enhanced engraftment of HPRT-knockdown anti-HIV-1 gene-modified cells (>2-fold, p < 0.0001) in humanized bone marrow/liver/thymus (huBLT) mice. Viral load was significantly reduced (>1 log fold, p < 0.001) in 6TG-treated HIV-1-infected huBLT mice compared to 6TG-untreated mice. We demonstrated that 6TG-mediated preconditioning and in vivo selection considerably improved engraftment of HPRT-knockdown anti-HIV-1 gene-modified HSPC and repopulation of anti-HIV-1 gene-modified hematopoietic cells in huBLT mice, allowing for efficient HIV-1 inhibition.
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Longitudinal clonal tracking in humanized mice reveals sustained polyclonal repopulation of gene-modified human-HSPC despite vector integration bias
(2021-12-01)
Suryawanshi, Gajendra W.
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Arokium, Hubert
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Kim, Sanggu
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Khamaikawin, Wannisa
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Lin, Samantha
Background: Current understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment. Methods: Humanized bone marrow-liver-thymus (hu-BLT) mice were generated by transplanting lentiviral vector-transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25 µl of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias. Results: A total of 897 HSPC clones were longitudinally tracked in hu-BLT mice—providing a first-ever demonstration of clonal dynamics and coordinated expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 (~ 60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within ± 1 kb of H3K36me3-enriched regions. Conclusions: Human repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.
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CRISPR/Cas9 genome editing of CCR5 combined with C46 HIV-1 fusion inhibitor for cellular resistant to R5 and X4 tropic HIV-1
(2024-12-01)
Khamaikawin, Wannisa
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Saisawang, Chonticha
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Tassaneetrithep, Boonrat
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Bhukhai, Kanit
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Phanthong, Phetcharat
Hematopoietic stem-cell (HSC) transplantation using a donor with a homozygous mutation in the HIV co-receptor CCR5 (CCR5Δ32/Δ32) holds great promise as a cure for HIV-1. Previously, there were three patients that had been reported to be completely cured from HIV infection by this approach. However, finding a naturally suitable Human Leukocyte Antigen (HLA)-matched homozygous CCR5Δ32 donor is very difficult. The prevalence of this allele is only 1% in the Caucasian population. Therefore, additional sources of CCR5Δ32/Δ32 HSCs are required. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one method to mediate CCR5 knockout in HSCs that has been successfully employed as a gene editing tool in clinical trials. Additional anti-HIV-1 strategies are still required for broad-spectrum inhibition of HIV-1 replication. Here in this study, we combined an additional anti-HIV-1 therapy, which is C46, a cell membrane-anchored HIV-1 fusion inhibitor with the CRISPR/Cas9 mediated knockout CCR5. The combined HIV-1 therapeutic genes were investigated for the potential prevention of both CCR5 (R5)- and CXCR4 (X4)-tropic HIV-1 infections in the MT4CCR5 cell line. The combinatorial CRISPR/Cas9 therapies were superior compared to single method therapy for achieving the HIV-1 cure strategy and shows potential for future applications.
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HIV-1 proviral DNA in purified peripheral blood CD34+ stem and progenitor cells in individuals with long-term HAART; paving the way to HIV gene therapy
(2024-02-29)
Tassaneetrithep, Boonrat
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Phuphuakrat, Angsana
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Pasomsub, Ekawat
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Bhukhai, Kanit
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Wongkummool, Wasinee
Human immunodeficiency virus (HIV)-1 infection is an important public health problem worldwide. After primary HIV-1 infection, transcribed HIV-1 DNA is integrated into the host genome, serving as a reservoir of the virus and hindering a definite cure. Although highly active antiretroviral therapy suppresses active viral replication, resulting in undetectable levels of HIV RNA in the blood, a viral rebound can be detected after a few weeks of treatment interruption. This supports the concept that there is a stable HIV-1 reservoir in people living with HIV-1. Recently, a few individuals with HIV infection were reported to be probably cured by hematopoietic stem transplantation (HSCT). The underlying mechanism for this success involved transfusion of uninfected hematopoietic stem and progenitor cells (HSPCs) from CCR5-mutated donors who were naturally resistant to HIV infection. Thus, gene editing technology to provide HIV-resistant HSPC has promise in the treatment of HIV infections by HSCT. In this study, we aimed to find HIV-infected individuals likely to achieve a definite cure via gene editing HSCT. We screened for total HIV proviral DNA by Alu PCR in peripheral blood mononuclear cells (PBMCs) of 20 HIV-infected individuals with prolonged viral suppression. We assessed the amount of intact proviral DNA via a modified intact proviral DNA assay (IPDA) in purified peripheral CD34+ HSPCs. PBMCs from all 20 individuals were positive for the gag gene in Alu PCR, and peripheral CD34+ HSPCs were IPDA-negative for six individuals. Our results suggested that these six HIV-infected individuals could be candidates for further studies into the ability of gene editing HSCT to lead to a definite HIV cure.
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Engineered Zinc Finger Protein Targeting 2LTR Inhibits HIV Integration in Hematopoietic Stem and Progenitor Cell-Derived Macrophages: In Vitro Study
(2022-02-01)
Chupradit, Koollawat
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Khamaikawin, Wannisa
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Sakkhachornphop, Supachai
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Puaninta, Chaniporn
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Torbett, Bruce E.
Human hematopoietic stem/progenitor cell (HSPC)-based gene therapy is a promising direction for curing HIV-1-infected individuals. The zinc finger protein (2LTRZFP) designed to target the 2-LTR-circle junction of HIV-1 cDNA was previously reported as an intracellular antiviral molecular scaffold that prevents HIV integration. Here, we elucidate the efficacy and safety of using 2LTRZFP in human CD34+ HSPCs. We transduced 2LTRZFP which has the mCherry tag (2LTRZFPmCherry) into human CD34+ HSPCs using a lentiviral vector. The 2LTRZFPmCherry-transduced HSPCs were subsequently differentiated into macrophages. The expression levels of pro-apoptotic proteins of the 2LTRZFPmCherry-transduced HSPCs showed no significant differ-ence from those of the non-transduced control. Furthermore, the 2LTRZFPmCherry-transduced HSPCs were successfully differentiated into mature macrophages, which had normal phagocytic function. The cytokine secretion assay demonstrated that 2LTRZFPmCherry-transduced CD34+ derived macrophages promoted the polarization towards classically activated (M1) subtypes. More importantly, the 2LTRZFPmCherry transduced cells significantly exhibited resistance to HIV-1 integration in vitro. Our findings demonstrate that the 2LTRZFPmCherry-transduced macrophages were found to be functionally and phenotypically normal, with no adverse effects of the anti-HIV-1 scaffold. Our data suggest that the anti-HIV-1 integrase scaffold is a promising antiviral molecule that could be applied to human CD34+ HSPC-based gene therapy for AIDS patients.
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Evaluation of a docetaxel-cisplatin-fluorouracil-Au complex in human oral carcinoma cell line
(2023-01-01)
Khamaikawin, Wannisa
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Locharoenrat, Kitsakorn
Higher tobacco and alcohol use have led to a consistent increase in head and neck cancer incidence rates. Currently employed chemotherapeutic and surgical treatment are associated with significant drawbacks. Herein, we evaluated the anti-tumour effect of gold nanoparticles as a vehicle for the delivery of a triple chemotherapy drug formulation and elucidated the potential underlying mechanism. The hydrodynamic size of docetaxel, cisplatin, and 5-fluorouracil physically co-adsorbed on Au nanoparticles was 56 ± 0.8 nm, showing a negative zeta potential. Fourier transform infra-red spectroscopy data confirmed that the triple chemotherapy drug successfully interacted with the gold nano-carrier. Au nanoparticles exhibited high loading efficacy of docetaxel (61%), cisplatin (75%), and 5-fluorouracil (90%), with a controlled drug release profile at 24 h. The triple chemotherapy drug formulation was tested in human oral cavity cancer cell line (KB). Cytotoxicity achieved through a synergistic effect between the treatments led to apoptosis, with a lower half-maximal inhibitory concentration indicating higher cytotoxicity than that of plain docetaxel-cisplatin-fluorouracil. Taken together, we demonstrated that the docetaxel-cisplatin-fluorouracil-gold complex exhibited excellent cytotoxicity in KB cells, superior to that docetaxel-cisplatin-fluorouracil.HIGHLIGHTS The docetaxel-cisplatin-fluorouracil-Au complex showed a controlled drug-release profile at 24 h. The docetaxel-cisplatin-fluorouracil-Au complex exhibited enhanced internalisation efficiency in cells. Au nanoparticles were biocompatible, with no change in apoptosis among cell line. Spherical Au nanoparticles allowed a high volume of incorporated docetaxel, cisplatin, and 5-fluorouracil to steadily attach onto cells.
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Mechanisms of virus dissemination in bone marrow of HIV-1–infected humanized BLT mice
(2019-10-01)
Ladinsky, Mark S.
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Khamaikawin, Wannisa
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Jung, Yujin
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Lin, Samantha
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Lam, Jennifer
Immune progenitor cells differentiate in bone marrow (BM) and then migrate to tissues. HIV-1 infects multiple BM cell types, but virus dissemination within BM has been poorly understood. We used light microscopy and electron tomography to elucidate mechanisms of HIV-1 dissemination within BM of HIV-1–infected BM/thymus/liver (BLT) mice. Tissue clearing combined with confocal and light sheet fluorescence microscopy revealed distinct populations of HIV-1 p24-producing cells in BM early after infection, and quantification of these populations identified macrophages as the principal subset of virus-producing cells in BM over time. Electron tomography demonstrated three modes of HIV-1 dissemination in BM: (i) semi-synchronous budding from T-cell and macrophage membranes, (ii) mature virus association with virus-producing T-cell uropods contacting putative target cells, and (iii) macrophages engulfing HIV-1–producing T-cells and producing virus within enclosed intracellular compartments that fused to invaginations with access to the extracellular space. These results illustrate mechanisms by which the specialized environment of the BM can promote virus spread locally and to distant lymphoid tissues.

Khamaikawin, Wannisa

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