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The cornea is the transparent outermost surface of the eye,consisting of a stratified epithelium,a collagenous stroma and an innermost single-cell layered endothelium and providing 2/3 of the refractive power of the eye. Multiple diseases of the cornea arise from genetic defects where the ultimate phenotype can be influenced by cross talk between the cell types and the extracellular matrix. Cell culture modeling of diseases can benefit from cornea organoids that include multiple corneal cell types and extracellular matrices. Here we present human iPS cell-derived organoids through sequential rounds of differentiation programs. These organoids share features of the developing cornea,harboring three distinct cell types with expression of key epithelial,stromal and endothelial cell markers. Cornea organoid cultures provide a powerful 3D model system for investigating corneal developmental processes and their disruptions in diseased conditions. View Publication

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study,we evaluated the efficacy of ∼200nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine),and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses,antigen-specific proliferation of peripheral blood mononuclear cells,gross and microscopic lung lesions,and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4+CD8$\alpha$$\alpha$+ T helper and CD8+ cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition,pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically,increased IFN-$\gamma$ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However,in the KAg nanovaccine-immunized pigs,hemagglutination inhibition,IgG and IgA antibody responses,and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall,our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs,with promise to induce cross-protective immunity. View Publication

Haematopoietic stem cell (HSC) gene therapy has demonstrated potential to treat many diseases. However,current state of the art requires sophisticated ex vivo gene transfer in a dedicated Good Manufacturing Practices facility,limiting availability. An automated process would improve the availability and standardized manufacture of HSC gene therapy. Here,we develop a novel program for semi-automated cell isolation and culture equipment to permit complete benchtop generation of gene-modified CD34+ blood cell products for transplantation. These cell products meet current manufacturing quality standards for both mobilized leukapheresis and bone marrow,and reconstitute human haematopoiesis in immunocompromised mice. Importantly,nonhuman primate autologous gene-modified CD34+ cell products are capable of stable,polyclonal multilineage reconstitution with follow-up of more than 1 year. These data demonstrate proof of concept for point-of-care delivery of HSC gene therapy. Given the many target diseases for gene therapy,there is enormous potential for this approach to treat patients on a global scale. View Publication

Acute myeloid leukemia (AML) is a heterogeneous group of aggressive bone marrow cancers arising from transformed hematopoietic stem and progenitor cells (HSPC). Therapy-related AML and MDS (t-AML/MDS) comprise a subset of AML cases occurring after exposure to alkylating chemotherapy and/or radiation and are associated with a very poor prognosis. Less is known about the pathogenesis and disease-initiating/leukemia stem cell (LSC) subpopulations of t-AML/MDS compared to their de novo counterparts. Here,we report the development of mouse models of t-AML/MDS. First,we modeled alkylator-induced t-AML/MDS by exposing wild type adult mice to N-ethyl-N-nitrosurea (ENU),resulting in several models of AML and MDS that have clinical and pathologic characteristics consistent with human t-AML/MDS including cytopenia,myelodysplasia,and shortened overall survival. These models were limited by their inability to transplant clinically aggressive disease. Second,we established three patient-derived xenograft models of human t-AML. These models led to rapidly fatal disease in recipient immunodeficient xenografted mice. LSC activity was identified in multiple HSPC subpopulations suggesting there is no canonical LSC immunophenotype in human t-AML. Overall,we report several new t-AML/MDS mouse models that could potentially be used to further define disease pathogenesis and test novel therapeutics. View Publication

Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58{\%} Venus(+) HSPCs with 6-16{\%} human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD),caused by mutations in the gp91phox subunit of the NADPH oxidase,TI of a gp91phox transgene into AAVS1 resulted in ∼15{\%} gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs,4-11{\%} of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases. View Publication

This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells),metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material contain the differentiated luminal and basal cell types,whereas organoids derived from prostate cancer tissue mimic the histology of the tumor. We explain how to establish these cultures in the fully defined serum-free conditioned medium that is required to sustain organoid growth. Starting with the plating of digested tissue material,full-grown organoids can usually be obtained in ∼2 weeks. The culture protocol we describe here is currently the only one that allows the growth of both the luminal and basal prostatic epithelial lineages,as well as the growth of advanced prostate cancers. Organoids established using this protocol can be used to study many different aspects of prostate biology,including homeostasis,tumorigenesis and drug discovery. View Publication

Immunotherapy has increased overall survival of metastatic cancer patients,and cancer antigens are promising vaccine targets. To fulfill the promise,appropriate tailoring of the vaccine formulations to mount in vivo cytotoxic T cell (CTL) responses toward co-delivered cancer antigens is essential. Previous development of therapeutic cancer vaccines has largely been based on studies in mice,and the majority of these candidate vaccines failed to induce therapeutic responses in the subsequent human clinical trials. Given that antigen dose and vaccine volume in pigs are translatable to humans and the porcine immunome is closer related to the human counterpart,we here introduce pigs as a supplementary large animal model for human cancer vaccine development. IDO and RhoC,both important in human cancer development and progression,were used as vaccine targets and 12 pigs were immunized with overlapping 20mer peptides spanning the entire porcine IDO and RhoC sequences formulated in CTL-inducing adjuvants: CAF09,CASAC,Montanide ISA 51 VG,or PBS. Taking advantage of recombinant swine MHC class I molecules (SLAs),the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1(*)04:01,-1(*)07:02,-2(*)04:01,-2(*)05:02,and/or -3(*)04:01. This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes. By IFN-$\gamma$ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses,and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior. These findings support the further use of pigs as a large animal model for vaccine development against human cancer. View Publication

The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D organoid system,we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes,including TMPRSS2-ERG fusion,SPOP mutation,SPINK1 overexpression,and CHD1 loss. Whole-exome sequencing shows a low mutational burden,consistent with genomics studies,but with mutations in FOXA1 and PIK3R1,as well as in DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies. View Publication

Small-cell lung cancer (SCLC),an aggressive neuroendocrine tumor with early dissemination and dismal prognosis,accounts for 15-20{\%} of lung cancer cases and ∼200,000 deaths each year. Most cases are inoperable,and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs),which are prevalent in SCLC,present a readily accessible 'liquid biopsy'. Here we show that CTCs from patients with either chemosensitive or chemorefractory SCLC are tumorigenic in immune-compromised mice,and the resultant CTC-derived explants (CDXs) mirror the donor patient's response to platinum and etoposide chemotherapy. Genomic analysis of isolated CTCs revealed considerable similarity to the corresponding CDX. Most marked differences were observed between CDXs from patients with different clinical outcomes. These data demonstrate that CTC molecular analysis via serial blood sampling could facilitate delivery of personalized medicine for SCLC. CDXs are readily passaged,and these unique mouse models provide tractable systems for therapy testing and understanding drug resistance mechanisms. View Publication