My research interest focuses on the gene regulation (NIBP, RGS4 and TCF4) and signal transduction (NFkB, protein trafficking and G protein signaling). I utilize innovative gene/cell therapy for the translational medicine to treat infectious/inflammatory diseases and genetic neurodevelopmental disorders. My overarching goal is to explore the translational application of improved CRISPR/Cas genome editors and develop innovative target cell-specific gene therapy in both preclinical animal studies and clinical trials. Also, I aim to elucidate the role and mechanism of NIBP, a novel protein I discovered(Hu et al. 2005), in regulating NFkB signaling and trans-Golgi network(Bodnar et al. 2020). NIBP may serve as a novel anti-viral restriction factor to eradicate/prevent virus infection (such as HIV, ZIKV, SARS-COV-2). Elucidating NIBP's functions may drive discovery and broad translational advances in neural development, adult neurogenesis, neuronal polarization, neuroregeneration, neuroinflammation, metabolic syndrome and tumorigenesis.I have established advantageous novel animal models, human induced pluripotent stem cells (iPSC)-derived vascularized microglia-containing cerebral organoids (vMCO) and CRISPR/Cas-mediated gene/cell therapy platform. I have the vision, expertise, and leadership skills to accomplish these goals. My creativity and productivity are reflected by my achievements during the past years.

·   Target-specific delivery of CRISPR/Cas (epi)genome editing and HIV cure

1. T cell-targeting gene therapy: HIV/AIDS remains a major public health concern, affecting >39 million people worldwide. In the era of antiretroviral treatment (ART), HIV remains incurable due to viral persistence and latency. HIV-1 infected individuals under long-term suppressive ART develop various comorbidities such as precocious aging, neurocognitive disorders (HAND), cardiovascular diseases, and others. ART does not eliminate the integrated and silent HIV-1 provirus in latently infected cells (CD4 T cells) and tissues, such as the lymphoid tissue, gastrointestinal/genital tract, bone marrow, or brain. Cessation of ART leads to rapid viral rebound, even after many years of viral suppression. Novel approaches to permanently silence or completely eliminate HIV-1 latent proviruses are urgently needed to achieve a “sterilizing” HIV cure. CRISPR/Cas genome editing strategy in preclinical animal studies including ours have shown a promise for eradicating HIV-1 provirus. Many viral and non-viral technologies have been developed for delivery of genome editors, which can be cDNA, mRNA, protein or ribonucleoprotein (RNP). RNP can be delivered via electroporation, nanoparticles (NP) or lentivirus-like particles (LVLP). In these projects, NP and VLP are used to deliver HIV-specific RNP because it does not involve the reverse transcription and random integration. Our approach involves CD4-targeted delivery of all-in-one Cas12f/multiplexed gRNA array for both in vitro and in vivo studies including humanized mice.
2. Microglia-targeted CRISPR/Cas editor for a cure of NeuroHIV: Although ART has greatly improved survival rates of HIV patients, >50% HIV-infected patients will still develop various degrees of HAND. Brain myeloid cells (BMC) including microglia (MG) and perivascular macrophages have been extensively investigated for their contribution to NeuroHIV persistence, chronic neuroinflammation and HAND. Thus, eradication of HIV provirus in MG is a critical step towards cure of NeuroHIV. Since AAV gene therapy is most promising with thousands of ongoing clinical trials, significant efforts to minimize the CRISPR/Cas editors for fitting into AAV size limit have identified several miniature Cas editors such as Cas12f, Cas12j and Cas13x. A scientific gap in this field is lack of ideal AAV serotypes (AAV-BM) with high efficiency of both crossing the BBB (AAV-B) and targeting MG (AAV-M) in vivo. The current AAV-B serotypes have high efficiency to transduce neurons, astrocytes, and oligodendrocytes, but have limited ability of transducing MG. In contrast, several AAV-M serotypes have been identified for their efficient tropism to MG in the literature and our preliminary studies. None of these AAV-M can cross BBB efficiently. Therefore, three strategies are employed to fill in this critical gap: 1) Developing novel Exo-ERVLP technologies for endogenous scissor gene delivery to MG via AAV-B sustained gene therapy; 2) Screening AAV-BM serotypes in non-human primate (NHP) for SIV eradication; and 3) Screening AAV-BM serotypes in humanized MG mouse model and vascularized MG-containing cerebral organoids (vMCO) for HIV eradication.

·   Regulation of NF-kB signaling pathways: NIBP.

NF-kB can be stimulated by many stimuli through various pathways.  It regulates an array of genes important in immunity, inflammation, stress response, cell growth and plasticity. High basal activity of NF-kB is reported in cancer cells, lymphocytes, neurons and smooth muscle cells. Sustained activation of NF-kB is critical for inflammation-linked cancer and other chronic diseases. However, the origins and mechanisms of NF-kB activation (constitutive or inducible) are not well understood. In most cases, NF-kB is held in the cytoplasm via Inhibitor of NF-kB (IkB). After phosphorylation by IkB kinase (IKK), IkB is degraded, leading to NF-kB activation. How IKK is activated remains a focus of considerable research interest.

My previous research interests were focused on the identification of novel proteins regulating IKK and its upstream kinases. Several novel proteins have been identified. Their functions and mechanisms need characterized. One of the novel proteins, NIBP, is shown to enhance TNFa and IL-1b-induced NF-kB activation via increasing IKK2 kinase activity and be required for growth and differentiation of neuronal cell line PC12 (Hu, et al., 2005). However, it is not known whether NIBP is essential for neuronal differentiation and survival in primary neurons or neural stem cells from central, peripheral and enteric nervous system. In addition, much more functions of NIBP and their mechanisms remain to be elucidated.

It is also a key member of trafficking protein particle (TRAPP) complex II (Cox et al., 2007; Kummel et al., 2008; Zong et al., 2011, 2012), implying its importance in regulating cellular trafficking. New clinical data showed that homozygous NIBP non-sense mutation is closely correlated with autosomal recessive mental retardation and neonatal microcephaly (Mir et al., 2009; Mochida et al., 2009; Philippe et al., 2009; Marangi et al., 2012). Homozygous deletion of NIBP genome in human leads to severe developmental delay, retinal dystrophy, hearing loss and dismorphic facial features (Koifman et al., 2010). Genome-wide assay identified two SNPs (single nucleotide polymorphisms) in NIBP that contribute to maternal effects on human height (Yoshida et al., 2010). NIBP is highly expressed in various human tumors. These clinical findings highlight the importance of NIBP in neurogenesis, mental development, neural functional integrity and tumorigenesis.

Hypothesis I: NIBP may play an important role in neurogenesis. The important role of NF-κB in neural development has been well established. In addition, NIBP may have some functions other than NF-κB regulation. Our preliminary data demonstrated that NIBP-like immunoreactivity is extensively expressed in neurons and neural stem/progenitor cells (including enteric nervous system).  To study the developmental role of NIBP, we generated conditional NIBP knockout or knockin mice using Cre/LoxP system. We also use Zebrafish neurogenic model and human induced pluripotent stem cells to study the role of NIBP signaling in neural induction and neurogenesis.

Hypothesis II: NIBP mediates neural regeneration after CNS (central nervous system) injury and disorders. The rationale is that regenerative failure in adult CNS may be partially due to the reduction or loss of NIBP expression after CNS injury. Lentivirus-mediated long-term and neuron-selective delivery of NIBP gene after adult CNS injury may promote nerve growth and ultimately improve functional recovery. We will first address the questions whether endogenous NIBP is required for central nerve growth and whether CNS injury reduces endogenous NIBP expression. Then, the lentivirus-mediated NIBP-shRNA strategy will be used to knock down the endogenous NIBP expression both in vitro and in vivo. Finally, lentivirus-mediated Cre/LoxP system will be employed to induce neuron-selective controllable expression of NIBP after CNS injury. Alternatively, neural stem/progenitor cells or other candidate cells infected with NIBP lentivirus will be transplanted into CNS after injury. The selective and inducible expression of NIBP will compensate for the reduction of NIBP after CNS injury and promote nerve growth through neuronal NFκB signaling while non-neuronal NFκB signaling will not be intervened.

Hypothesis III:  NIBP regulates tumorigenesis. Unigene assay indicated that NIBP EST (Expression Sequence Tag) is widely present in various human tumors. Gene Expression Omnibus (GEO) profiles from many microarray experiments suggested that NIBP is highly expressed in cancer cells. The preliminary studies demonstrated that NIBP is selectively expressed in various cancer cell lines, and a similar interaction between endogenous NIBP and IKK2 is present in MCF7 and HCT116 cells. Immunohistochmistry and TissueScan real-time PCR analysis showed that NIBP is highly expressed in various human tumors. IKK2 is critical in regulating NF-kB activation and tumorigenesis. IKK2-specific inhibitors have been targeted for therapeutic development and clinical application. Thus, NIBP highly expressed in tumor cells may retain the constitutive activation of IKK2 and contribute to the proliferation, tumorigenesis, metastasis and drug-resistance of cancer cells. The expression pattern of NIBP in a number of cancer cell lines and human tumors will be profiled. In vivo studies using nude mice and cell-specific conditional NIBP knockout mice will be performed. The mechanisms for NIBP regulation and function in tumorigenesis will be investigated. Results from these studies will not only establish the novel role of NIBP in cancer development and progression but also provide an important insight into the mechanism of IKK2-mediated activation of NF-kB in tumor cells. NIBP may represent a novel adaptor protein or regulator that controls the activities of IKK complex and NF-kB.

Hypothesis IV:  Hypothalamic NIBP signaling in neurodevelopmental disordes and obesity. Autosomal recessive mutations of the gene NIBP (TRAPPC9) have been linked to a novel developmental disorder known as NIBP syndrome. Some of the phenotypical features reported in patients include moderate-to-severe intellectual disability, microencephaly, facial dysmorphia, hypotonia, autism spectrum disorder. Importantly, obesity has been reported in over half of clinical cases, although this number is likely higher as not all studies addressed this phenotype. To model NIBP syndrome, we have developed 4 lines of NIBP knockout/knockin mice. Global NIBP knockout mice developed obesity, hyperphagia, increased fat mass, and impaired glucose/insulin tolerance, even under a normal diet. Furthermore, hypothalamus-specific NIBP knockout mice also spontaneously developed obesity. This highlighted the role for hypothalamic NIBP in maintaining energy homeostasis. The on-going projects utilize vMCO from iPSCs of patients with NIBP syndrome as well as several innovative mouse models to explore the role of NIBP in hypothalamic neurogenesis and obesity pathogenesis.

·   Regulation of gastrointestinal motility and inflammation by NIBP/NFκB signaling.

·   Transcription factor 4 regulates spinogenesis and synaptogenesis
The proneural proteins of the class I/II basic-helix-loop-helix (bHLH) family are highly conserved transcription factors. Class I bHLH proteins are expressed in a broad number of tissues during development, whereas class II bHLH protein expression is more tissue restricted. Current understanding of the function of class I/II bHLH transcription factors in both invertebrate and vertebrate neurobiology is largely focused on their function as regulators of neurogenesis. TCF4 mutations have been reliably identified in genome-wide association studies as a susceptibility risk factor for schizophrenia and have also been associated with Pitt-Hopkins syndrome, Fuchs’ endothelial corneal dystrophy, and primary sclerosing. It is unknown whether these diseases are due to defects in neurogenesis, in mature differentiated cells, or both. My research interest aims to decipher the cytoplasmic functions of Tcf4 in regulating spinogenesis and synaptogenesis of postmitotic neurons.