S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection.

Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting 'anchoring' non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.

Intra-hematopoietic cell fusion as a source of somatic variation in the hematopoietic system.

Cell fusion plays a well-recognized, physiological role during development. Bone-marrow-derived hematopoietic cells have been shown to fuse with non-hematopoietic cells in a wide variety of tissues. Some organs appear to resolve the changes in ploidy status, generating functional and mitotically-competent events. However, cell fusion exclusively involving hematopoietic cells has not been reported. Indeed, genomic copy number variation in highly replicative hematopoietic cells is widely considered a hallmark of malignant transformation. Here we show that cell fusion occurs between cells of the hematopoietic system under injury as well as non-injury conditions. Experiments reveal the acquisition of genetic markers in fusion products, their tractable maintenance during hematopoietic differentiation and long-term persistence after serial transplantation. Fusion events were identified in clonogenic progenitors as well as differentiated myeloid and lymphoid cells. These observations provide a new experimental model for the study of non-pathogenic somatic diversity in the hematopoietic system.

Estrogen receptor, progesterone receptor, interleukin-6 and interleukin-8 are variable in breast cancer and benign stem/progenitor cell populations.

BACKGROUND: Estrogen receptor positive breast cancers have high recurrence rates despite tamoxifen therapy. Breast cancer stem/progenitor cells (BCSCs) initiate tumors, but expression of estrogen (ER) or progesterone receptors (PR) and response to tamoxifen is unknown. Interleukin-6 (IL-6) and interleukin-8 (IL-8) may influence tumor response to therapy but expression in BCSCs is also unknown. METHODS: BCSCs were isolated from breast cancer and benign surgical specimens based on CD49f/CD24 markers. CD44 was measured. Gene and protein expression of ER alpha, ER beta, PR, IL-6 and IL-8 were measured by proximity ligation assay and qRT-PCR. RESULTS: Gene expression was highly variable between patients. On average, BCSCs expressed 10-106 fold less ERα mRNA and 10-103 fold more ERß than tumors or benign stem/progenitor cells (SC). BCSC lin-CD49f-CD24-cells were the exception and expressed higher ERα mRNA. PR mRNA in BCSCs averaged 10-104 fold less than in tumors or benign tissue, but was similar to benign SCs. ERα and PR protein detection in BCSCs was lower than ER positive and similar to ER negative tumors. IL-8 mRNA was 10-104 higher than tumor and 102 fold higher than benign tissue. IL-6 mRNA levels were equivalent to benign and only higher than tumor in lin-CD49f-CD24-cells. IL-6 and IL-8 proteins showed overlapping levels of expressions among various tissues and cell populations. CONCLUSIONS: BCSCs and SCs demonstrate patient-specific variability of gene/protein expression. BCSC gene/protein expression may vary from that of other tumor cells, suggesting a mechanism by which hormone refractory disease may occur.

FDA Approval Summary: Capmatinib and Tepotinib for the Treatment of Metastatic NSCLC Harboring MET Exon 14 Skipping Mutations or Alterations.

The FDA approved capmatinib and tepotinib on May 6, 2020, and February 3, 2021, respectively. Capmatinib is indicated for patients with metastatic non-small cell lung cancer (mNSCLC) whose tumors have a mutation leading to mesenchymal-epithelial transition (MET) exon 14 skipping as detected by an FDA-approved test. Tepotinib is indicated for mNSCLC harboring MET exon 14 skipping alterations. The approvals were based on trials GEOMETRY mono-1 (capmatinib) and VISION (tepotinib). In GEOMETRY mono-1, overall response rate (ORR) per Blinded Independent Review Committee (BIRC) was 68% [95% confidence interval (CI), 48-84] with median duration of response (DoR) 12.6 months (95% CI, 5.5-25.3) in 28 treatment-naïve patients and 41% (95% CI: 29, 53) with median DoR 9.7 months (95% CI, 5.5-13) in 69 previously treated patients with NSCLC with mutations leading to MET exon 14 skipping. In VISION, ORR per BIRC was 43% (95% CI: 32, 56) with median DoR 10.8 months (95% CI, 6.9-not estimable) in 69 treatment-naïve patients and 43% (95% CI, 33-55) with median DoR 11.1 months (95% CI, 9.5-18.5) in 83 previously-treated patients with NSCLC harboring MET exon 14 alterations. These are the first two therapies to be FDA approved specifically for patients with metastatic NSCLC with MET exon 14 skipping.

FDA Approval Summary: Selpercatinib for the Treatment of Lung and Thyroid Cancers with RET Gene Mutations or Fusions.

On May 8, 2020, the FDA granted accelerated approval to selpercatinib for (i) adult patients with metastatic RET fusion-positive non-small cell lung cancer (NSCLC), (ii) adult and pediatric patients ≥12 years of age with advanced or metastatic RET-mutant medullary thyroid cancer who require systemic therapy, and (iii) adult and pediatric patients ≥12 years of age with advanced or metastatic RET fusion-positive thyroid cancer who require systemic therapy and who are radioactive iodine refractory (if radioactive iodine is appropriate). Approval was granted on the basis of the clinically important effects on the overall response rate (ORR) with prolonged duration of responses observed in a multicenter, open-label, multicohort clinical trial (LIBRETTO-001, NCT03157128) in patients whose tumors had RET alterations. ORRs within the approved patient populations ranged from 64% [95% confidence interval (CI), 54-73] in prior platinum-treated RET fusion-positive NSCLC to 100% (95% CI, 63-100) in systemic therapy-naïve RET fusion-positive thyroid cancer, with the majority of responders across indications demonstrating responses of at least 6 months. The product label includes warnings and precautions for hepatotoxicity, hypertension, QT interval prolongation, hemorrhagic events, hypersensitivity, risk of impaired wound healing, and embryo-fetal toxicity. This is the first approval of a drug specifically for patients with RET alterations globally.

Entry kinetics and cell-cell transmission of surface-bound retroviral vector particles.

BACKGROUND: Transduction with recombinant HIV-1 derived lentivirus vectors is a multi-step process initiated by surface attachment and subsequent receptor-directed uptake into the target cell. We previously reported the retention of vesicular stomatitis virus G protein pseudotyped particles on murine progenitor cells and their delayed cell-cell transfer. METHODS: To examine the underlying mechanism in more detail, we used a combination of approaches focused on investigating the role of receptor-independent factors in modulating attachment. RESULTS: The investigation of synchronized transduction reveals cell-type specific rates of vector particle clearance with substantial delays during particle entry into murine hematopoietic progenitor cells. The observed uptake kinetics from the surface of the 1 degrees cell correlate inversely with the magnitude of transfer to 2 degrees targets, corresponding with our initial observation of preferential cell-cell transfer in the context of brief vector exposures. We further demonstrate that vector particle entry into cells is associated with the cell-type specific abundance of extracellular matrix fibronectin. Residual particle-extracellular fibronectin matrix binding and 2 degrees transfer can be competitively disrupted by heparin exposure without affecting murine progenitor homing and repopulation. CONCLUSIONS: Although cellular attachment factors, including fibronectin, aid gene transfer by colocalizing particles to cells and disfavoring early dissociation from targets, they also appear to stabilize particles on the cell surface. The present study highlights the inadvertent consequences for cell entry and cell-cell transfer.

CXCR4 but not CXCR7 is mainly implicated in ocular leukocyte trafficking during ovalbumin-induced acute uveitis.

Uveitis is an inflammatory ocular disease characterized by the infiltration of T lymphocytes and other leukocytes into the eye. The recruitment of these inflammatory cells from systemic vasculature to ocular tissue is a well-coordinated multistep process including rolling, firm adhesion and transmigration. CXCL12 (SDF-1alpha) is an endothelial cell-derived cytokine interacting with CXCR4 and CXCR7, two chemokine receptors mainly expressed in T cells, neutrophils and monocytes. Recent studies have shown that CXCR4, CXCR7 and their ligand, CXCL12, are important for the regulation of leukocyte mobilization and trafficking. However, it is unclear whether these two chemokine receptors are implicated in the pathogenesis of uveitis. In this study, we used DO11.10 mice, whose CD4+ T cells are genetically engineered to react with ovalbumin (OVA), to investigate the role of CXCR4 and CXCR7 in an animal model of uveitis. Intravital microscopy revealed that intravitreal OVA challenge of DO11.10 mice caused the infiltration of both T cells and neutrophils. The invasion of these inflammatory cells coincided with the detection of transcriptional up-regulation of CXCR4 and CXCR7 in the eye. In addition, both real-time-PCR and immunohistochemistry revealed an enhanced expression of endothelial CXCL12. Furthermore, intraperitoneal injection of AMD3100 (a specific CXCR4 antagonist) significantly attenuated OVA-induced uveitis and CXCL12-mediated transwell migration. In contrast, intraperitoneal administration of CXCR7 neutralizing antibody did not significantly alter ocular infiltration of inflammatory cells caused by OVA challenge. Our data suggest that CXCR4 but not CXCR7 plays a critical role in antigen-induced ocular inflammation by facilitating leukocyte infiltration. This study not only enhances our knowledge of the immunopathological mechanism of uveitis but also provides a novel rationale to target CXCR4 as an anti-inflammatory strategy to treat uveitis.

CXCR4 induction in hematopoietic progenitor cells from Fanca(-/-), -c(-/-), and -d2(-/-) mice.

OBJECTIVE: Bone marrow failure is a near-universal occurrence in patients with Fanconi anemia (FA) and is thought to result from exhaustion of the hematopoietic stem cell (HSC) pool. Retrovirus-mediated expression of the deficient protein corrects this phenotype and makes FA a candidate disease for HSC-directed gene therapy. However, inherent repopulation deficits and stem cell attrition during conventional transduction culture prevent therapeutic chimerism. MATERIALS AND METHODS: We previously reported rapid transduction protocols to limit stem cell losses after ex vivo culture. Here we describe a complementary strategy intended to improve repopulation through upregulation of chemokine receptor (CXCR) 4, a principal factor in hematopoietic homing. RESULTS: Using murine models with transgenic disruption of Fanca, -c, and -d2, we found that c-kit(+) and sca-1(+) progenitor cells express levels of CXCR4 comparable with those of wild-type littermates. Lineage-depleted progenitor populations rapidly upregulated CXCR4 transcript and protein in response to cytokine stimulation or hypoxia, regardless of genotype. Hypoxia conditioning of lineage-depleted Fancc(-/-) progenitors also reduced oxidative stress, improved in vitro migration and led to improved chimerism in myeloablated recipients after transplantation. CONCLUSION: These studies provide evidence that CXCR4 regulation in progenitor cells from transgenic mice representing multiple FA genotypes is intact and that modulation of homing offers a potential strategy to offset the FA HSC repopulation deficiency.

The frequency of CC to TT tandem mutations in mismatch repair-deficient cells is increased in a cytosine run.

Mononucleotide runs are hot spots for frameshift mutations in mismatch repair (MMR)-deficient cells. However, a role for mononucleotide runs in the formation of base pair substitutions has not been tested. Previously, we demonstrated that ultraviolet radiation C (UVC)- or reactive oxygen species-induced CC to TT tandem mutations are markedly enhanced in MMR-deficient cells. The target for the mutational analysis was two cytosines in a run of five cytosines (5C) within mouse Aprt. Because mutation from C to T for either or both of the two critical cytosines created a codon yielding a functional Aprt protein, this assay allowed both single and tandem substitutions to be quantified and the relative ratios compared. To determine if the cytosine run increased the frequency of single and/or tandem base pair substitutions, alternative constructs were created in which the cytosine run was disrupted by flanking the target cytosines with either thymines (2Cpyr) or adenines (2Cpur). Disruption of the cytosine run dramatically decreased the frequency of UVC-induced tandem mutations in the 2Cpyr and 2Cpur constructs, as compared with the 5C construct. Moreover, CC to TT tandem mutations occurred spontaneously or were induced by oxidative stress only within the 5C construct. These results demonstrate that CC to TT tandem mutations in MMR-deficient cells form more readily in a homocytosine run than in a sequence limited to two cytosines.