Generation of humanized mouse models to support therapeutic development for SYNGAP1 and STXBP1 disorders.

Heterozygous variants in SYNGAP1 and STXBP1 lead to distinct neurodevelopmental disorders caused by haploinsufficient levels of post-synaptic SYNGAP1 and pre-synaptic STXBP1, which are critical for normal synaptic function. While several gene-targeted therapeutic approaches have proven efficacious in vitro, these often target regions of the human gene that are not conserved in rodents, hindering the pre-clinical development of these compounds and their transition to the clinic. To overcome this limitation, here we generate and characterize Syngap1 and Stxbp1 humanized mouse models in which we replaced the mouse Syngap1 and Stxbp1 gene, respectively, with the human counterpart, including regulatory and non-coding regions. Fully humanized Syngap1 mice present normal viability and can be successfully crossed with currently available Syngap1 haploinsufficiency mouse models to generate Syngap1 humanized haploinsufficient mice. Stxbp1 mice were successfully humanized, yet exhibit impaired viability (particularly males) and reduced STXBP1 protein abundance. Mouse viability could be improved by outcrossing this model to other mouse strains, while Stxbp1 humanized females and hybrid mice can be used to evaluate target engagement of human-specific therapeutics. Overall, these humanized mouse models represent a broadly available tool to further pre-clinical therapeutic development for SYNGAP1 and STXBP1 disorders.

Oleuropein mediated autophagy begets antimalarial drug resistance.

Drug resistance in Plasmodium falciparum presents a formidable challenge to the humanity. And, unavailability of an effective vaccine worsens the situation further. Autophagy is one of the mechanisms employed by parasite to evade drug pressure to survive. Autophagy induced by the P. falciparum in response to the oleuropein pressure may answer many questions related to the parasite survival as well as evolving drug tolerance. The survival/autophagy axis could be an important avenue to explore in order to address certain questions related to the evolution of drug resistance. In addition, humanized mouse model of P. falciparum infection could serve as an important preclinical tool to investigate the oleuropein-induced autophagy, potentially helping to dissect the mechanisms underlying the development of antimalarial drug resistance.

PBMC-engrafted humanized mice models for evaluating immune-related and anticancer drug delivery systems.

Immune-related drug delivery systems (DDSs) in humanized mouse models are at the forefront of cancer research and serve as bridges between preclinical studies and clinical applications. These systems offer unique platforms for exploring new therapies and understanding their interactions with human cells and the immune system. Here, we focus on a DDS and a peripheral blood mononuclear cell (PBMC)-engrafted humanized mouse model that we recently developed, and consider some of the key components, challenges, and applications to advance these systems towards better cancer treatment on the basis of a better understanding of the immune response. Our DDS is unique and has a dual function, an anticancer effect and a capacity to fine-tune the immune reaction. The PBL-NOG-hIL-4-Tg mouse system is superior to other available humanized mouse systems for the development of such multifunctional DDSs because it supports the rapid reconstruction of an individual donor's immunity and avoids the onset of graft-versus-host disease.

Development of New Diffuse Large B Cell Lymphoma Mouse Models.

Diffuse large B cell lymphoma (DLBCL) is the most diagnosed, aggressive non-Hodgkin lymphoma, with ~40% of patients experiencing refractory or relapsed disease. Given the low response rates to current therapy, alternative treatment strategies are necessary to improve patient outcomes. Here, we sought to develop an easily accessible new xenograft mouse model that better recapitulates the human disease for preclinical studies. We generated two Luciferase (Luc)-EGFP-expressing human DLBCL cell lines representing the different DLBCL cell-of-origin subtypes. After intravenous injection of these cells into humanized NSG mice, we monitored the tumor growth and evaluated the organ-specific engraftment/progression period. Our results showed that human IL6-expressing NSG (NSG-IL6) mice were highly permissive for DLBCL cell growth. In NSG-IL6 mice, systemic engraftments of both U2932 activated B cell-like- and VAL germinal B cell-like-DLBCL (engraftment rate; 75% and 82%, respectively) were detected within 2nd-week post-injection. In the organ-specific ex vivo evaluation, both U2932-Luc and VAL-Luc cells were initially engrafted and expanded in the spleen, liver, and lung and subsequently in the skeleton, ovary, and brain. Consistent with the dual BCL2/MYC translocation association with poor patient outcomes, VAL cells showed heightened proliferation in human IL6-conditioned media and caused rapid tumor expansion and early death in the engrafted mice. We concluded that the U2932 and VAL cell-derived human IL6-expressing mouse models reproduced the clinical features of an aggressive DLBCL with a highly consistent pattern of tumor development. Based on these findings, NSG mice expressing human IL6 have the potential to serve as a new tool to develop DLBCL xenograft models to overcome the limitations of standard subcutaneous DLBCL xenografts.

CRISPR-mediated megabase-scale transgene de-duplication to generate a functional single-copy full-length humanized DMD mouse model.

BACKGROUND: The development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes. RESULTS: To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout. CONCLUSIONS: Our unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.

CCL19-producing fibroblasts promote tertiary lymphoid structure formation enhancing anti-tumor IgG response in colorectal cancer liver metastasis.

Tertiary lymphoid structures (TLSs) are associated with enhanced immunity in tumors. However, their formation and functions in colorectal cancer liver metastasis (CRLM) remain unclear. Here, we reveal that intra- and peri-tumor mature TLSs (TLS+) are associated with improved clinical outcomes than TLS- tumors. Using single-cell-RNA-sequencing and spatial-enhanced-resolution-omics-sequencing (Stereo-seq), we reveal that TLS+ tumors are enriched with IgG+ plasma cells (PCs), while TLS- tumors are characterized with IgA+ PCs. By generating TLS-associated PC-derived monoclonal antibodies in vitro, we show that TLS-PCs secrete tumor-targeting antibodies. As the proof-of-concept, we demonstrate the anti-tumor activities of TLS-PC-mAb6 antibody in humanized mouse model of colorectal cancer. We identify a fibroblast lineage secreting CCL19 that facilitates lymphocyte trafficking to TLSs. CCL19 treatment promotes TLS neogenesis and prevents tumor growth in mice. Our data uncover the central role of CCL19+ fibroblasts in TLS formation, which in turn generates therapeutic antibodies to restrict CRLM.

Modeling HBV Infection and Therapy in Immunodeficient NOD-Rag1-/-IL2RgammaC-null (NRG) Fumarylacetoacetate Hydrolase (FAH) Knockout Mice with Human Chimeric Liver.

Chimeric mouse models with a humanized liver (Hu-HEP mice) provide a unique tool to study human hepatotropic virus diseases, including viral infection, viral pathogenesis, and anti-viral therapy. Here, we describe a detailed protocol for studying hepatitis B infection in NRG-derived fumarylacetoacetate hydrolase (FAH) knockout mice repopulated with human hepatocytes (FRG-Hu HEP mice). The procedures include (1) maintenance and genotyping of the FRG mice, (2) intrasplenic injection of primary human hepatocytes (PHH), (3) 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) drug reduction cycling to improve human hepatocyte repopulation, (4) human albumin detection, and (5) HBV infection and detection. The method is simple and allows for highly reproducible generation of FRG-Hu HEP mice for HBV infection and therapy investigations.

Establishment of a humanized mouse model using steady-state peripheral blood-derived hematopoietic stem and progenitor cells facilitates screening of cancer-targeted T-cell repertoires.

Background: Cancer-targeted T-cell receptor T (TCR-T) cells hold promise in treating cancers such as hematological malignancies and breast cancers. However, approaches to obtain cancer-reactive TCR-T cells have been unsuccessful. Methods: Here, we developed a novel strategy to screen for cancer-targeted TCR-T cells using a special humanized mouse model with person-specific immune fingerprints. Rare steady-state circulating hematopoietic stem and progenitor cells were expanded via three-dimensional culture of steady-state peripheral blood mononuclear cells, and then the expanded cells were applied to establish humanized mice. The human immune system was evaluated according to the kinetics of dendritic cells, monocytes, T-cell subsets, and cytokines. To fully stimulate the immune response and to obtain B-cell precursor NAML-6- and triple-negative breast cancer MDA-MB-231-targeted TCR-T cells, we used the inactivated cells above to treat humanized mice twice a day every 7 days. Then, human T cells were processed for TCR ß-chain (TRB) sequencing analysis. After the repertoires had been constructed, features such as the fraction, diversity, and immune signature were investigated. Results: The results demonstrated an increase in diversity and clonality of T cells after treatment. The preferential usage and features of TRBV, TRBJ, and the V-J combination were also changed. The stress also induced highly clonal expansion. Tumor burden and survival analysis demonstrated that stress induction could significantly inhibit the growth of subsequently transfused live tumor cells and prolong the survival of the humanized mice. Conclusions: We constructed a personalized humanized mouse model to screen cancer-targeted TCR-T pools. Our platform provides an effective source of cancer-targeted TCR-T cells and allows for the design of patient-specific engineered T cells. It therefore has the potential to greatly benefit cancer treatment.

Technical considerations and strategies for generating and optimizing humanized mouse tumor models in immuno-oncology research.

The field of cancer immunotherapy has experienced significant progress, resulting in the emergence of numerous biological drug candidates requiring in vivo efficacy testing and a better understanding of their mechanism of action (MOA). Humanized immune system (HIS) models are valuable tools in this regard. However, there is a lack of systematic guidance on HIS modeling. To address this issue, the present study aimed to establish and optimize a variety of HIS models for immune-oncology (IO) study, including genetically engineered mouse models and HIS models with human immune components reconstituted in severely immunocompromised mice. The efficacy and utility of these models were tested with several marketed or investigational IO drugs according to their MOA, followed by immunophenotypic analysis and efficacy evaluation. The results of the present study demonstrated that the HIS models responded to various IO drugs as expected and that each model had unique niches, utilities and limitations. Researchers should carefully choose the appropriate models based on the MOA and the targeted immune cell populations of the investigational drug. The present study provides valuable methodologies and actionable technical guidance on designing, generating or utilizing appropriate HIS models to address specific questions in translational IO.

Design, synthesis, and evaluation of antitumor activity of 2-arylmethoxy-4-(2-fluoromethyl-biphenyl-3-ylmethoxy) benzylamine derivatives as PD-1/PD-l1 inhibitors.

A series of novel 2-arylmethoxy-4-(2-fluoromethyl-biphenyl-3-ylmethoxy) benzylamine derivatives was designed, synthesized, and evaluated for their antitumor effects as PD-1/PD-L1 inhibitors both in vitro and in vivo. Firstly, the ability of these compounds to block the PD-1/PD-L1 immune checkpoint was assessed using the homogeneous time-resolved fluorescence (HTRF) assay. Two of the compounds can strongly block the PD-1/PD-L1 interaction, with IC50 values of less than 10 nM, notably, compound HD10 exhibited significant clinical potential by inhibiting the PD-1/PD-L1 interaction with an IC50 value of 3.1 nM. Further microscale thermophoresis (MST) analysis demonstrated that HD10 had strong interaction with PD-L1 protein. Co-crystal structure (2.7 Å) analysis of HD10 in complex with the PD-L1 protein revealed a strong affinity between the compound and the target PD-L1 dimer. This provides a solid theoretical basis for further in vitro and in vivo studies. Next, a typical cell-based experiment demonstrated that HD10 could remarkably prevent the interaction of hPD-1 293 T cells from human recombinant PD-L1 protein, effectively restoring T cell function, and promoting IFN-γ secretion in a dose-dependent manner. Moreover, HD10 was effective in suppressing tumor growth (TGI = 57.31 %) in a PD-1/PD-L1 humanized mouse model without obvious toxicity. Flow cytometry, qPCR, and immunohistochemistry data suggested that HD10 inhibits tumor growth by activating the immune system in vivo. Based on these results, it seems likely that HD10 is a promising clinical candidate that should be further investigated.

Peripheral CD8+PD-1+ T cells as novel biomarker for neoadjuvant chemoimmunotherapy in humanized mice of non-small cell lung cancer.

Neoadjuvant immunotherapy has shown promising clinical activity in the treatment of early non-small cell lung cancer (NSCLC); however, further clarification of the specific mechanism and identification of biomarkers are imperative prior to implementing it as a daily practice. The study investigated the reprogramming of T cells in both tumor and peripheral blood following neoadjuvant chemoimmunotherapy in a preclinical NSCLC mouse model engrafted with a human immune system. Samples were also collected from 21 NSCLC patients (Stage IA-IIIB) who received neoadjuvant chemoimmunotherapy, and the dynamics of potential biomarkers within these samples were measured and further subjected to correlation analysis with prognosis. Further, we initially investigated the sources of the potential biomarkers. We observed in the humanized mouse model, neoadjuvant chemoimmunotherapy could prevent postoperative recurrence and metastasis by increasing the frequency and cytotoxicity of CD8+ T cells in both peripheral blood (p < 0.001) and tumor immune microenvironment (TIME) (p < 0.001). The kinetics of peripheral CD8+PD-1+ T cells reflected the changes in the TIME and pathological responses, ultimately predicting survival outcome of mice. In the clinical cohort, patients exhibiting an increase in these T cells post-treatment had a higher rate of complete or major pathological response (p < 0.05) and increased immune infiltration (p = 0.0012, r = 0.792). We identified these T cells originating from tumor draining lymph nodes and subsequently entering the TIME. In conclusion, the kinetics of peripheral CD8+PD-1+ T cells can serve as a predictor for changes in TIME and optimal timing for surgery, ultimately reflecting the outcomes of neoadjuvant chemoimmunotherapy in both preclinical and clinical setting.

Kaposi sarcoma-associated herpesvirus complement control protein (KCP) and glycoprotein K8.1 are not required for viral infection in vitro or in vivo.

Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causal agent of Kaposi sarcoma, a cancer that appears as tumors on the skin or mucosal surfaces, as well as primary effusion lymphoma and KSHV-associated multicentric Castleman disease, which are B-cell lymphoproliferative disorders. Effective prophylactic and therapeutic strategies against KSHV infection and its associated diseases are needed. To develop these strategies, it is crucial to identify and target viral glycoproteins involved in KSHV infection of host cells. Multiple KSHV glycoproteins expressed on the viral envelope are thought to play a pivotal role in viral infection, but the infection mechanisms involving these glycoproteins remain largely unknown. We investigated the role of two KSHV envelope glycoproteins, KSHV complement control protein (KCP) and K8.1, in viral infection in various cell types in vitro and in vivo. Using our newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP, K8.1, or both, we demonstrated the presence of KCP and K8.1 on the surface of both virions and KSHV-infected cells. We showed that KSHV lacking KCP and/or K8.1 remained infectious in KSHV-susceptible cell lines, including epithelial, endothelial, and fibroblast, when compared to wild-type recombinant KSHV. We also provide the first evidence that KSHV lacking K8.1 or both KCP and K8.1 can infect human B cells in vivo in a humanized mouse model. Thus, these results suggest that neither KCP nor K8.1 is required for KSHV infection of various host cell types and that these glycoproteins do not determine KSHV cell tropism. IMPORTANCE: Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic human gamma-herpesvirus associated with the endothelial malignancy Kaposi sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman disease. Determining how KSHV glycoproteins such as complement control protein (KCP) and K8.1 contribute to the establishment, persistence, and transmission of viral infection will be key for developing effective anti-viral vaccines and therapies to prevent and treat KSHV infection and KSHV-associated diseases. Using newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP and/or K8.1, we show that KCP and K8.1 can be found on the surface of both virions and KSHV-infected cells. Furthermore, we show that KSHV lacking KCP and/or K8.1 remains infectious to diverse cell types susceptible to KSHV in vitro and to human B cells in vivo in a humanized mouse model, thus providing evidence that these viral glycoproteins are not required for KSHV infection.

In vivo adenine base editing rescues adrenoleukodystrophy in a humanized mouse model.

X-linked adrenoleukodystrophy (ALD), an inherited neurometabolic disorder caused by mutations in ABCD1, which encodes the peroxisomal ABC transporter, mainly affects the brain, spinal cord, adrenal glands, and testes. In ALD patients, very-long-chain fatty acids (VLCFAs) fail to enter the peroxisome and undergo subsequent ß-oxidation, resulting in their accumulation in the body. It has not been tested whether in vivo base editing or prime editing can be harnessed to ameliorate ALD. We developed a humanized mouse model of ALD by inserting a human cDNA containing the pathogenic variant into the mouse Abcd1 locus. The humanized ALD model showed increased levels of VLCFAs. To correct the mutation, we tested both base editing and prime editing and found that base editing using ABE8e(V106W) could correct the mutation in patient-derived fibroblasts at an efficiency of 7.4%. Adeno-associated virus (AAV)-mediated systemic delivery of NG-ABE8e(V106W) enabled robust correction of the pathogenic variant in the mouse brain (correction efficiency: ∼5.5%), spinal cord (∼5.1%), and adrenal gland (∼2%), leading to a significant reduction in the plasma levels of C26:0/C22:0. This established humanized mouse model and the successful correction of the pathogenic variant using a base editor serve as a significant step toward treating human ALD disease.

A novel humanized mouse model for HIV and tuberculosis co-infection studies.

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to be a major public health problem worldwide. The human immunodeficiency virus (HIV) is another equally important life-threatening pathogen. HIV infection decreases CD4+ T cell levels markedly increasing Mtb co-infections. An appropriate animal model for HIV/Mtb co-infection that can recapitulate the diversity of the immune response in humans during co-infection would facilitate basic and translational research in HIV/Mtb infections. Herein, we describe a novel humanized mouse model. Methods: The irradiated NSG-SGM3 mice were transplanted with human CD34+ hematopoietic stem cells, and the humanization was monitored by staining various immune cell markers for flow cytometry. They were challenged with HIV and/or Mtb, and the CD4+ T cell depletion and HIV viral load were monitored over time. Before necropsy, the live mice were subjected to pulmonary function test and CT scan, and after sacrifice, the lung and spleen homogenates were used to determine Mtb load (CFU) and cytokine/chemokine levels by multiplex assay, and lung sections were analyzed for histopathology. The mouse sera were subjected to metabolomics analysis. Results: Our humanized NSG-SGM3 mice were able to engraft human CD34+ stem cells, which then differentiated into a full-lineage of human immune cell subsets. After co-infection with HIV and Mtb, these mice showed decrease in CD4+ T cell counts overtime and elevated HIV load in the sera, similar to the infection pattern of humans. Additionally, Mtb caused infections in both lungs and spleen, and induced granulomatous lesions in the lungs. Distinct metabolomic profiles were also observed in the tissues from different mouse groups after co-infections. Conclusion: The humanized NSG-SGM3 mice are able to recapitulate the pathogenic effects of HIV and Mtb infections and co-infection at the pathological, immunological and metabolism levels and are therefore a reproducible small animal model for studying HIV/Mtb co-infection.

Generation of Anti-HIV CAR-T Cells for Preclinical Research.

The inability of people living with HIV (PLWH) to eradicate human immunodeficiency virus (HIV) infection is due in part to the inadequate HIV-specific cellular immune response. The antiviral function of cytotoxic CD8+ T cells, which are crucial for HIV control, is impaired during chronic viral infection because of viral escape mutations, immune exhaustion, HIV antigen downregulation, inflammation, and apoptosis. In addition, some HIV-infected cells either localize to tissue sanctuaries inaccessible to CD8+ T cells or are intrinsically resistant to CD8+ T cell killing. The novel design of synthetic chimeric antigen receptors (CARs) that enable T cells to target specific antigens has led to the development of potent and effective CAR-T cell therapies. While initial clinical trials using anti-HIV CAR-T cells performed over 20 years ago showed limited anti-HIV effects, the improved CAR-T cell design, which enabled its success in treating cancer, has reinstated CAR-T cell therapy as a strategy for HIV cure with notable progress being made in the recent decade.Effective CAR-T cell therapy against HIV infection requires the generation of anti-HIV CAR-T cells with potent in vivo activity against HIV-infected cells. Preclinical evaluation of anti-HIV efficacy of CAR-T cells and their safety is fundamental for supporting the initiation of subsequent clinical trials in PLWH. For these preclinical studies, we developed a novel humanized mouse model supporting in vivo HIV infection, the development of viremia, and the evaluation of novel HIV therapeutics. Preclinical assessment of anti-HIV CAR-T cells using this mouse model involves a multistep process including peripheral blood mononuclear cells (PBMCs) harvested from human donors, T cell purification, ex vivo T cell activation, transduction with lentiviral vectors encoding an anti-HIV CAR, CAR-T cell expansion and infusion in mice intrasplenically injected with autologous PBMCs followed by the determination of CAR-T cell capacity for HIV suppression. Each of the steps described in the following protocol were optimized in the lab to maximize the quantity and quality of the final anti-HIV CAR-T cell products.

Correction of human nonsense mutation via adenine base editing for Duchenne muscular dystrophy treatment in mouse.

Duchenne muscular dystrophy (DMD) is the most prevalent herediatry disease in men, characterized by dystrophin deficiency, progressive muscle wasting, cardiac insufficiency, and premature mortality, with no effective therapeutic options. Here, we investigated whether adenine base editing can correct pathological nonsense point mutations leading to premature stop codons in the dystrophin gene. We identified 27 causative nonsense mutations in our DMD patient cohort. Treatment with adenine base editor (ABE) could restore dystrophin expression by direct A-to-G editing of pathological nonsense mutations in cardiomyocytes generated from DMD patient-derived induced pluripotent stem cells. We also generated two humanized mouse models of DMD expressing mutation-bearing exons 23 or 30 of human dystrophin gene. Intramuscular administration of ABE, driven by ubiquitous or muscle-specific promoters could correct these nonsense mutations in vivo, albeit with higher efficiency in exon 30, restoring dystrophin expression in skeletal fibers of humanized DMD mice. Moreover, a single systemic delivery of ABE with human single guide RNA (sgRNA) could induce body-wide dystrophin expression and improve muscle function in rotarod tests of humanized DMD mice. These findings demonstrate that ABE with human sgRNAs can confer therapeutic alleviation of DMD in mice, providing a basis for development of adenine base editing therapies in monogenic diseases.

A humanized knock-in Col6a1 mouse recapitulates a deep-intronic splice-activating variant.

Antisense therapeutics such as splice-modulating antisense oligonucleotides (ASOs) are promising tools to treat diseases caused by splice-altering intronic variants. However, their testing in animal models is hampered by the generally poor sequence conservation of the intervening sequences between human and other species. Here we aimed to model in the mouse a recurrent, deep-intronic, splice-activating, COL6A1 variant, associated with a severe form of Collagen VI-related muscular dystrophies (COL6-RDs), for the purpose of testing human-ready antisense therapeutics in vivo. The variant, c.930+189C>T, creates a donor splice site and inserts a 72-nt-long pseudoexon, which, when translated, acts in a dominant-negative manner, but which can be skipped with ASOs. We created a unique humanized mouse allele (designated as "h"), in which a 1.9 kb of the mouse genomic region encoding the amino-terminus (N-) of the triple helical (TH) domain of collagen a1(VI) was swapped for the human orthologous sequence. In addition, we also created an allele that carries the c.930+189C>T variant on the same humanized knock-in sequence (designated as "h+189T"). We show that in both models, the human exons are spliced seamlessly with the mouse exons to generate a chimeric mouse-human collagen a1(VI) protein. In homozygous Col6a1 h+189T/h+189T mice, the pseudoexon is expressed at levels comparable to those observed in heterozygous patients' muscle biopsies. While Col6a1h/h mice do not show any phenotype compared to wildtype animals, Col6a1 h/h+189T and Col6a1 h+189T/h+189T mice have smaller muscle masses and display grip strength deficits detectable as early as 4 weeks of age. The pathogenic h+189T humanized knock-in mouse allele thus recapitulates the pathogenic splicing defects seen in patients' biopsies and allows testing of human-ready precision antisense therapeutics aimed at skipping the pseudoexon. Given that the COL6A1 N-TH region is a hot-spot for COL6-RD variants, the humanized knock-in mouse model can be utilized as a template to introduce other COL6A1 pathogenic variants. This unique humanized mouse model thus represents a valuable tool for the development of antisense therapeutics for COL6-RDs.

PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice.

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

A Novel Humanized Mouse Model for HIV and Tuberculosis Co-infection Studies.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to be a major public health problem worldwide. The human immunodeficiency virus (HIV) is another equally important life-threatening pathogen. Further, co-infections with HIV and Mtb have severe effects in the host, with people infected with HIV being fifteen to twenty-one times more likely to develop active TB. The use of an appropriate animal model for HIV/Mtb co-infection that can recapitulate the diversity of the immune response in humans would be a useful tool for conducting basic and translational research in HIV/Mtb infections. The present study was focused on developing a humanized mouse model for investigations on HIV-Mtb co-infection. Using NSG-SGM3 mice that can engraft human stem cells, our studies showed that they were able to engraft human CD34+ stem cells which then differentiate into a full-lineage of human immune cell subsets. After co-infection with HIV and Mtb, these mice showed decrease in CD4+ T cell counts overtime and elevated HIV load in the sera, similar to the infection pattern of humans. Additionally, Mtb caused infections in both lungs and spleen, and induced the development of granulomatous lesions in the lungs, detected by CT scan and histopathology. Distinct metabolomic profiles were also observed in the tissues from different mouse groups after co-infections. Our results suggest that the humanized NSG-SGM3 mice are able to recapitulate the effects of HIV and Mtb infections and co-infection in the human host at pathological, immunological and metabolism levels, providing a dependable small animal model for studying HIV/Mtb co-infection.

A novel GARP humanized mouse model for efficacy assessment of GARP-targeting therapies.

Although breakthroughs have been achieved with immune checkpoint inhibitors (ICI) therapy, some tumors do not respond to those therapies due to primary or acquired resistance. GARP, a type I transmembrane cell surface docking receptor mediating latent transforming growth factor-ß (TGF-ß) and abundantly expressed on regulatory T lymphocytes and platelets, is a potential target to render these tumors responsive to ICI therapy, and enhancing anti-tumor response especially combined with ICI. To facilitate these research efforts, we developed humanized mouse models expressing humanized GARP (hGARP) instead of their mouse counterparts, enabling in vivo assessment of GARP-targeting agents. We created GARP-humanized mice by replacing the mouse Garp gene with its human homolog. Then, comprehensive experiments, including expression analysis, immunophenotyping, functional assessments, and pharmacologic assays, were performed to characterize the mouse model accurately. The Tregs and platelets in the B-hGARP mice (The letter B is the first letter of the company's English name, Biocytogen.) expressed human GARP, without expression of mouse GARP. Similar T, B, NK, DCs, monocytes and macrophages frequencies were identified in the spleen and blood of B-hGARP and WT mice, indicating that the humanization of GARP did not change the distribution of immune cell in these compartments. When combined with anti-PD-1, monoclonal antibodies (mAbs) against GARP/TGF-ß1 complexes demonstrated enhanced in vivo anti-tumor activity compared to monotherapy with either agent. The novel hGARP model serves as a valuable tool for evaluating human GARP-targeting antibodies in immuno-oncology, which may enable preclinical studies to assess and validate new therapeutics targeting GARP. Furthermore, intercrosses of this model with ICI humanized models could facilitate the evaluation of combination therapies.