Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies.

Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression.

Tumor-infiltrating mast cells are associated with resistance to anti-PD-1 therapy.

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34 cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγ (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression.

Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations.

Human acute myeloid leukemia blast-derived exosomes in patient-derived xenograft mice mediate immune suppression.

Exosomes are virus-size membrane-bound vesicles of endocytic origin present in all body fluids. Plasma of AML patients is significantly enriched in exosomes, which carry a cargo of immunosuppressive molecules and deliver them to recipient immune cells, suppressing their functions. However, whether these exosomes originate from leukemic blasts or from various normal cells in the bone marrow or other tissues is unknown.

Human tumor-associated monocytes/macrophages and their regulation of T cell responses in early-stage lung cancer.

Data from mouse tumor models suggest that tumor-associated monocyte/macrophage lineage cells (MMLCs) dampen antitumor immune responses. However, given the fundamental differences between mice and humans in tumor evolution, genetic heterogeneity, and immunity, the function of MMLCs might be different in human tumors, especially during early stages of disease. Here, we studied MMLCs in early-stage human lung tumors and found that they consist of a mixture of classical tissue monocytes and tumor-associated macrophages (TAMs).

Cytokines increase engraftment of human acute myeloid leukemia cells in immunocompromised mice but not engraftment of human myelodysplastic syndrome cells.

Patient-derived xenotransplantation models of human myeloid diseases including acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms are essential for studying the biology of the diseases in pre-clinical studies. However, few studies have used these models for comparative purposes. Previous work has shown that acute myeloid leukemia blasts respond to human hematopoietic cytokines whereas myelodysplastic syndrome cells do not.

Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge.

Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles.

Pharmacologic inhibition of the Menin-MLL interaction blocks progression of MLL leukemia in vivo.

Chromosomal translocations affecting mixed lineage leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report the development of highly potent and orally bioavailable small-molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, and show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia.

Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells.

Many patients with acute myeloid leukemia (AML) are incurable with chemotherapy and may benefit from novel approaches. One such approach involves the transfer of T cells engineered to express chimeric antigen receptors (CARs) for a specific cell-surface antigen. This strategy depends upon preferential expression of the target on tumor cells.

The potent oncogene NPM-ALK mediates malignant transformation of normal human CD4(+) T lymphocytes.

With this study we have demonstrated that in vitro transduction of normal human CD4(+) T lymphocytes with NPM-ALK results in their malignant transformation. The transformed cells become immortalized and display morphology and immunophenotype characteristic of patient-derived anaplastic large-cell lymphomas. These unique features, which are strictly dependent on NPM-ALK activity and expression, include perpetual cell growth, proliferation, and survival; activation of the key signal transduction pathways STAT3 and mTORC1; and expression of CD30 (the hallmark of anaplastic large-cell lymphoma) and of immunosuppressive cytokine IL-10 and cell-surface protein PD-L1/CD274.

Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection.

HIV-1 entry into CD4(+) T cells requires binding of the virus to CD4 followed by engagement of either the C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor 4 (CXCR4) coreceptor. Pharmacologic blockade or genetic inactivation of either coreceptor protects cells from infection by viruses that exclusively use the targeted coreceptor. We have used zinc-finger nucleases to drive the simultaneous genetic modification of both ccr5 and cxcr4 in primary human CD4(+) T cells.

Novel recombinant human b7-h4 antibodies overcome tumoral immune escape to potentiate T-cell antitumor responses.

B7-H4 (VTCN1, B7x, B7s) is a ligand for inhibitory coreceptors on T cells implicated in antigenic tolerization. B7-H4 is expressed by tumor cells and tumor-associated macrophages (TAM), but its potential contributions to tumoral immune escape and therapeutic targeting have been less studied. To interrogate B7-H4 expression on tumor cells, we analyzed fresh primary ovarian cancer cells collected from patient ascites and solid tumors, and established cell lines before and after in vivo passaging.

Mitochondrial energetic and AKT status mediate metabolic effects and apoptosis of metformin in human leukemic cells.

Previous reports demonstrate that metformin, an anti-diabetic drug, can decrease the risk of cancer and inhibit cancer cell growth. However, its mechanism in cancer cells is still unknown. Metformin significantly blocks cell cycle and inhibits cell proliferation and colony formation of leukemic cells.

Maintenance of hematopoietic stem cells through regulation of Wnt and mTOR pathways.

Hematopoietic stem cell (HSC) self renewal and lineage commitment depend on complex interactions with the microenvironment. The ability to maintain or expand HSCs for clinical applications or basic research has been substantially limited because these interactions are not well defined. Recent evidence suggests that HSCs reside in a low-perfusion, reduced-nutrient niche and that nutrient-sensing pathways contribute to HSC homeostasis.

Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases.

HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains.

TAT-mediated transduction of NF-Ya peptide induces the ex vivo proliferation and engraftment potential of human hematopoietic progenitor cells.

Retroviral overexpression of NF-Ya, the regulatory subunit of the transcription factor NF-Y, activates the transcription of multiple genes implicated in hematopoietic stem cell (HSC) self-renewal and differentiation and directs HSCs toward self-renewal. We asked whether TAT-NF-Ya fusion protein could be used to transduce human CD34(+) cells as a safer, more regulated alternative approach to gene therapy. Here we show that externally added recombinant protein was able to enter the cell nucleus and activate HOXB4, a target gene of NF-Ya, using real-time polymerase chain reaction RNA and luciferase-based protein assays.

Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo.

Persistence of T cells engineered with chimeric antigen receptors (CARs) has been a major barrier to use of these cells for molecularly targeted adoptive immunotherapy. To address this issue, we created a series of CARs that contain the T cell receptor-zeta (TCR-zeta) signal transduction domain with the CD28 and/or CD137 (4-1BB) intracellular domains in tandem. After short-term expansion, primary human T cells were subjected to lentiviral gene transfer, resulting in large numbers of cells with >85% CAR expression.

Umbilical cord blood xenografts in immunodeficient mice reveal that T cells enhance hematopoietic engraftment beyond overcoming immune barriers by stimulating stem cell differentiation.

Clinical experience and animal models have shown that donor T cell depletion (TCD) adversely affects engraftment of hematopoietic stem cells (HSCs). Although it is known that donor T cells are acting to overcome residual host immune barriers, they may also exert effects independent of host resistance via direct or indirect interactions with donor stem cells, their microenvironment, or key differentiation events. To more precisely define the effect of T cells on engraftment, we have performed human umbilical cord blood (UCB) transplantation into immunodeficient mice under limiting dilution conditions.

Immunosurveillance and survivin-specific T-cell immunity in children with high-risk neuroblastoma.

Purpose: Tumor immunosurveillance influences oncogenesis and tumor growth, but it remains controversial whether clinical failure of immunosurveillance is a result of lymphocyte dysfunction or tumor escape. In this study, our goal was to characterize the physiology of tumor immunosurveillance in children with high-risk neuroblastoma (HR-NBL).

Patients And Methods: Immunohistopathologic studies were carried out on 26 tumor samples from a cohort of HR-NBL patients diagnosed at Children's Hospital of Philadelphia for the 2-year period from May 2003 to May 2005.