Gene therapy lacks tools to deliver in a safe and tissue-targeted manner. Limitations include toxicity, off-target effects due to targeting, and inability to repeat dose. We have developed a novel megakaryocyte-derived extracellular vesicle (MkEV)-based non-viral gene therapy delivery platform that preferentially targets bone marrow in vivo. Here we present work showing that STRM.BIO MkEVs: 1) pass through the liver and spleen to preferentially target bone marrow in mice and non-human primates (NHPs); 2) can selectively deliver pDNA cargo to long term HSCs to drive reporter protein expression exclusively in bone marrow following intravenous delivery in mice; and 3) present a platform to develop and deliver targeted gene therapies in vivo that are safe for repeat dosing. Our vision is to open the door to the future of medicine for patients living with rare diseases worldwide and bring gene therapy to life. Primary human CD34+ hematopoietic stem and progenitor cells (hHSPCs) were differentiated into megakaryocytes in vitro. MkEVs were harvested and loaded with pDNA via electroporation, followed by DNase treatment to remove free cargo. For in vivo biodistribution and cargo-mediated protein expression studies, STRM.BIO MkEVs were exogenously labeled with fluorescent dye (PKH26, Cell Tracker Deep Red, and/or DiD), loaded with cargo, and IV-injected into mouse tail veins (wild type or NSG) or NHPs (cynomolgus monkeys). Tissues were collected 16-48h after IV injection, with MkEV tissue biodistribution quantified by dye fluorescence (Mean Fluorescence Intensity (MFI), plate reader) normalized to tissue weight or genomic DNA input, and pDNA cargo quantified by qPCR. Additionally, quantification of MkEVs+ cells was performed by flow cytometry analysis of murine bone marrow derived sub-populations (lineage negative, c-Kit +, Sca-1 + (LSK); lineage positive (Lin +); and, long-term HSCs (LT-HSCs; Lin -/CD150 +/CD201 +)) cells. Protein expression from MkEV-loaded pDNA cargo was similarly determined by plate reader fluorescence data and confocal microscopy. In mouse biodistribution studies, DiD-labeled MkEVs were predominantly detected in bone marrow, followed by liver and spleen 16h-post tail vein injection (plate reader analyses: Bone Marrow - 1.1e 4 ± 0.2 SD; Liver - 9.6e 3 ± 1.0 SD; Spleen - 1.8e 4 ± 0.2 SD. MFI/g tissue; n=3mice/group). Flow cytometry analyses show MkEVs preferentially targeted the hematopoietic compartment (of the MkEV+ cells, 94 ± 1% CD45+ vs 6 ± 1% CD45-, respectively), specifically, the HSPCs (68%, LSK) and LT-HSCs (100%) 48h post intravenous delivery via tail vein injection. Pharmacokinetic studies show that while MkEVs can circulate through highly vascularized tissues like the liver and spleen, they accumulate in the bone marrow. Strikingly, miRFP encoded by the pDNA was exclusively detected in bone marrow (p<0.0001 vs. all other tissues; Fig. 1). In NHPs, dye-labeled MkEVSs were loaded with a MkEV-driven eGFP reporter plasmid. Studies showed preferential biodistribution to bone marrow, and preferential qPCR amplification of the pDNA cargo in bone marrow (both MFI/ ACTB and GFP/ ACTB were significantly higher vs. all other tissues: range p<0.05 through p<0.0001), Fig. 2. A tolerability and repeat dosing study was performed in NHPs. Monkeys were injected with 4 weekly doses of STRM.BIO MkEVs and serial bloodwork was obtained 6h post dose for cytokine evaluation and 5 days post dose for end organ damage assessment (n=4). The study showed no evidence of kidney, hematologic, or liver damage as evidenced by normal creatinine and electrolytes, no significant changes in complete blood counts, coagulation parameters, or liver function tests. There was no evidence of significant cytokine release or inflammatory changes on tissue histology. These data confirm that MkEVs are non-toxic and the platform is amenable to repeat dosing. Taken together, these data establish an advanced gene therapy platform that can target bone marrow specifically following in vivo administration in both mice and NHPs, potentially eliminating the need for current ex vivo approaches to treat rare blood disorders, and providing the critical ability to repeat dose as required in clinic. STRM.BIO is leveraging our platform to develop gene therapies for rare blood diseases in people.

"Comparison of Transplantation of Lung Organoid Cell Types: One Size Does Not Fit All." American Journal of Respiratory Cell and Molecular Biology, 66(3), pp. 340–343

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34(+) hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34(+) cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.

Heterozygous inactivating mutations in ribosomal protein genes (RPGs) are associated with hematopoietic and developmental abnormalities, activation of p53, and altered risk of cancer in humans and model organisms. Here we performed a large‐scale analysis of cancer genome data to examine the frequency and selective pressure of RPG lesions across human cancers. We found that hemizygous RPG deletions are common, occurring in about 43 Hemizygous deletion of ribosomal protein genes (RPGs) is a strikingly common vulnerability of human cancers that associates with TP53 mutations and could be targetable therapeutically. Hemizygous deletion of ribosomal protein genes (RPGs) is a strikingly common vulnerability of human cancers that associates with TP53 mutations and could be targetable therapeutically.

Ribosomal protein (RP) mutations are found in many diseases, including Diamond Blackfan anemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancer risk are major complications. RP mutations are thought to cause p53 activation through accumulation of free RPs that bind and sequester MDM2, the negative regulator of p53. We previously characterized a zebrafish mutant in rps29, a ribosomal gene found mutated in DBA patients. Rps29-/- embryos have hematopoietic and endothelial defects, including decreased cmyb and flk1 expression and defects in hemoglobinization. Consistent with other animal models of RP dysfunction, p53 knockdown in rps29-/- embryos rescued these defects. To uncover novel compounds that correct the phenotypes of DBA, we performed a chemical screen in rps29-/- embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued hemoglobin (Hb) levels in the mutant embryo. To confirm that CaM inhibitors could rescue mammalian models of DBA, we tested them in human and murine models. Treating cord blood-derived CD34+ cells deficient in RPS19, the CaM inhibitor trifluoperazine (TFP) relieved the erythroid differentiation block. Injection of TFP in a DBA murine model significantly increased red blood cell number and Hb levels and reduced p53 activity in the bone marrow. Of note, the effect of TFP was specific to RP deficiency and had no effect on erythroid differentiation or p53 activity in WT cells or mice. This prompted us to hypothesize that TFP is selectively blocking signaling in the RP deficient state, possibly through inhibition of CaM-dependent kinases. In vitro kinase profiling of over 100 kinases revealed that TFP and other positive hits from our screen inhibited the activity of p70 ribosomal S6 kinase (p70S6K) and multiple members of p90 ribosomal S6 kinase (RSK) family. RSKs are highly conserved serine/threonine kinases that regulate cell growth, migration, and survival. They can activate mTOR and directly phosphorylate RPS6. Strikingly, RSK protein levels are elevated in lysates from rps29-/- embryos and treatment with RSK or p70S6K inhibitors increased Hb in rps29-/- embryos in vivo, mimicking CaM inhibitors. RSK phosphorylation is also increased in human RPS19-deficient peripheral blood-derived CD34+ cells compared to WT cells. Treatment with TFP reduced RSK phosphorylation and decreased signaling downstream of RSK, including p70S6K, but only in the presence of RP deficiency. Similarly, TFP reduced p53 and phosphorylation of p53 at S392, but only in the presence of RP deficiency. An in vitro kinase assay determined that p70S6K directly phosphorylated p53 at S392 but not other commonly modified residues, S15 and S20. Mass spectrometry analysis of posttranslational modifications of p53 isolated from TFP treated cells revealed a significant reduction of peptides that contain phosphorylated S392. We hypothesized that TFP inhibited phosphorylation of S392. To test this hypothesis, phosphomimetic mutants were transfected into Saos2 cells and p53 transcriptional activity was evaluated using p21mRNA levels. TFP treatment of cells containing WT p53 or a negative control transactivation domain mutant, p53-S15D, resulted in a 4-fold reduction in p21 mRNA levels, while cells containing p53-S392D had no reduction in p21 mRNA in response to TFP. In conclusion, we have shown that RP deficiency increases RSK phosphorylation and CaM inhibitors decrease signaling downstream of RSK, which leads to a reduction of p53 activity and rescues the phenotypes of multiple in vitro and in vivo models of DBA. Our data strongly suggests that CaM inhibitors may be effective therapies for DBA patients, and a clinical trial is being planned with TFP for 2017.

Ribosomal protein (RP) mutations are found in many diseases, including Diamond Blackfan anemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancer risk are major complications. RP mutations cause p53 activation through accumulation of free RPs that bind and sequester MDM2, the negative regulator of p53. We previously characterized a zebrafish mutant in rps29 , a gene found mutated in DBA patients. Rps29 -/- embryos have hematopoietic and endothelial defects, including decreased c myb and flk1 expression and defects in hemoglobinization. Consistent with other animal models of RP dysfunction, p53 knockdown in rps29-/- embryos rescued these defects. To uncover novel compounds that correct the phenotypes of DBA, we performed a chemical screen in rps29-/- embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued hemoglobin (Hb) levels in the mutant embryo. To confirm that CaM inhibitors could rescue mammalian models of DBA, we applied them to human and murine models. Treating cord blood-derived CD34+ cells deficient in RPS19 with the CaM inhibitor, trifluoperazine (TFP), relieved the erythroid differentiation block. Injection of TFP in a DBA murine model significantly increased red blood cell number and Hb levels. Mechanistic studies in A549 cells infected with lentivirus expressing RPS19 shRNA demonstrated that TFP blocks p53 nuclear accumulation and induction of multiple p53 transcriptional target genes ( p <0.05). Through p53 genetic manipulation, we determined that TFP inhibits p53 transcriptional activity through its c-terminal domain (CTD). Since this region has many residues that can be phosphorylated by CaM-dependent kinases, we hypothesized that TFP blocked phosphorylation of residues in the CTD. To test this hypothesis, phosphomimetic mutants were transfected into Saos2 cells and p53 transcriptional activity in response to TFP was evaluated using p21mRNA levels. TFP treatment of cells containing WT p53 or a transactivation domain mutant, S15D, resulted in a 4-fold reduction in p21 mRNA levels, while all four phosphomimetic mutants in the CTD had attenuated responses to TFP (<2-fold). The common CaM-dependent kinases that phosphorylate these CTD residues are Chk1 and Chk2. Investigation of the role of Chk1 and Chk2 found that a chk2 morpholino and multiple inhibitors of Chk2, but not Chk1, rescued Hb levels in the rps29-/- embryo ( p <0.05). Chk2 inhibitors also mimic CaM inhibition in our in vitro assays. In conclusion, we have shown a novel mechanism by which CaM inhibitors mediate p53 activity through the CTD and can rescue the phenotypes of multiple in vitro and in vivo models of DBA. Our data strongly suggests that CaM or Chk2 inhibitors may be effective therapies for DBA patients, and a clinical trial is being planned with TFP.Disclosures Ebert: Genoptix: Consultancy, Patents & Royalties; H3 Biomedicine: Consultancy; Celgene: Consultancy. Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Ribosomopathies such as 5q- syndrome and Diamond-Blackfan anemia are broadly characterized by haploinsufficiency of one or more ribosome-associated proteins leading to an aberrant activation of the p53 pathway. The prevailing hypothesis for the mechanism of p53 stabilization and activation in the context of ribosomal protein haploinsufficiency suggests that increased binding of a subset of free ribosomal proteins (including RPL5, RPL11, RPL23a, and RPS7) to MDM2 abrogates the negative regulatory control of MDM2 on p53. In order to understand more fully how the binding partner profile of MDM2 changes as a consequence of ribosome dysfunction, we performed a SILAC-based IP/MS proteomics screen on MDM2 immunoprecipitates to identify proteins that were differentially bound to MDM2 with and without shRNA-mediated knockdown of RPS14, a commonly haploinsufficient gene in 5q- syndrome. A V5-tagged MDM2 was expressed in A549 cells, and lentivirally-delivered shRNAs were expressed that targeted either luciferase (for control) or RPS14. Immunoprecipitations were performed on whole cell lysates using a V5 antibody and analyzed by mass spectrometry to identify peptides associated with MDM2 in each condition. We found that RPL5, RPL11, and RPL23a were strongly associated with MDM2 in the context of RPS14 knockdown, but were also abundant in MDM2 immunoprecipitates when a control hairpin was used. Another ribosomal protein, RPL38, was also strongly associated with MDM2 in both conditions. In contrast, RPS14 knockdown appeared to abrogate binding of RPS7 to MDM2, which was strongly associated in the control knockdown condition only. These data corroborate previous findings that a subset of 60S subunit ribosomal proteins can physically associate with MDM2, and they further suggest that this binding may commonly occur even in the absence of ribosomal haploinsufficiency. While differential binding of free ribosomal proteins to MDM2 may indeed contribute to p53 pathway activation in ribosomopathies, these data suggest that the difference in binding is subtle and may be a part of a larger set of p53-activating signals. Additional non-ribosomal proteins were identified that were specifically associated with MDM2 in the context of ribosomal protein deficiency, and follow-up experiments are ongoing to interrogate the biological consequences of these context-specific interactions.

Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner.

More than a decade has passed since the initial identification of ribosomal protein gene mutations in patients with Diamond-Blackfan anemia (DBA), a hematologic disorder that became the founding member of a class of diseases known as ribosomopathies. In these diseases, genetic abnormalities that result in defective ribosome biogenesis cause strikingly tissue-specific phenotypes in patients, specifically bone marrow failure, craniofacial abnormalities and skeletal defects. Several animal models and numerous in vitro studies have demonstrated that the p53 pathway is central to the ribosomopathy phenotype. Additionally, there is mounting evidence of a link between the dysregulation of components of the translational machinery and the pathology of various malignancies. The importance of the role of ribosomal dysfunction in the pathogenesis of hematologic disorders is becoming clearer, and elucidation of the underlying mechanisms could have broad implications for both basic cellular biology and clinical intervention strategies.

Abstract Abstract 512 Ribosomal protein mutations are common in patients with Diamond Blackfan anemia (DBA), who have red cell aplasia and craniofacial abnormalities, and rps14 deficiency has been linked to 5q- myelodysplastic syndrome. We have characterized a zebrafish mutant in rps29, a ribosomal protein in the small subunit. Rps29−/− embryos have hematopoietic and endothelial defects, including decreased hematopoietic stem cells, defects in hemoglobinization, and decreased staining of vessel markers. Heads of mutant embryos are also morphologically affected, with increased apoptosis. Consistent with other models of DBA, knockdown of p53 completely rescues the rps29 hematopoietic and apoptotic phenotypes. We wanted to identify chemicals that could rescue the rps29 mutant phenotype. Using 600 compounds from known bioactive libraries, we performed an in vivo chemical screen for rescue of the rps29 apoptotic and endothelial defects. Treatment with one compound, A-3, improved the morphology of the apoptotic embryo head. A-3 is an inhibitor of calmodulin, which interacts with a host of calmodulin-dependent enzymes and can bind to other proteins in the cell, including p21. W-7, another calmodulin inhibitor related to A-3, can rescue the endothelial defect in the rps29 mutant. Calmodulin inhibition in rps29 mutant embryos rescues the hemoglobin defect. Furthermore, structurally unrelated chemicals that inhibit calmodulin rescue the endothelial defect. We hypothesized that calmodulin inhibition was affecting some part of the p53 pathway. We tested whether A-3 had an effect on the phenotype in vitro in a cancer cell line where shRNA knockdown of RPS19 has been shown to stabilize p53 and induce p21. When rps19 is knocked down in A549 cells, treatment with calmodulin inhibitors resulted in a decrease of p21 protein, as measured by flow cytometry, although p53 protein levels were stable. This effect was also observed in primary human cord blood-derived CD34+ hematopoietic stem and progenitor cells. In situ immunofluorescence in A549s further revealed that nuclear localization of p53 and p21 upon RPS19 knockdown was disrupted by chemical treatment. Calmodulin inhibitors also rescue ribosomal protein deficiency-mediated erythroid defects both in vitro and in vivo. When RPS19 is knocked down in CD34+ cells, there is a defect in erythroid differentiation. Treatment with calmodulin inhibitors rescued production of CD71+ cells upon differentiation. In conclusion, we have demonstrated the use of calmodulin inhibitors as a novel approach for mediating p53 activation upon ribosomal protein knockdown, thereby rescuing the erythroid defects of ribosomal deficiency. Our data suggests that small molecules that inhibit calmodulin may be effective therapies for patients with ribosomal deficiency. Disclosures: Ebert: Celgene: Consultancy; Genoptix: Consultancy. Zon:Fate Therapeutics: Founder Other.

Bronchopulmonary dysplasia (BPD) remains a major complication of prematurity resulting in significant morbidity and mortality. The pathology of BPD is multifactorial and leads to alveolar simplification and distal lung injury. Previous studies have shown a beneficial effect of systemic treatment with bone marrow-derived mesenchymal stromal cells (MSCs) and MSC-conditioned media (MSC-CM) leading to amelioration of the lung parenchymal and vascular injury in vivo in the hyperoxia murine model of BPD. It is possible that the beneficial response from the MSCs is at least in part due to activation of endogenous lung epithelial stem cells. Bronchioalveolar stem cells (BASCs) are an adult lung stem cell population capable of self-renewal and differentiation in culture, and BASCs proliferate in response to bronchiolar and alveolar lung injury in vivo. Systemic treatment of neonatal hyperoxia-exposed mice with MSCs or MSC-CM led to a significant increase in BASCs compared with untreated controls. Treatment of BASCs with MSC-CM in culture showed an increase in growth efficiency, indicating a direct effect of MSCs on BASCs. Lineage tracing data in bleomycin-treated adult mice showed that Clara cell secretory protein-expressing cells including BASCs are capable of contributing to alveolar repair after lung injury. MSCs and MSC-derived factors may stimulate BASCs to play a role in the repair of alveolar lung injury found in BPD and in the restoration of distal lung cell epithelia. This work highlights the potential important role of endogenous lung stem cells in the repair of chronic lung diseases.

Abstract Abstract 3479 Diseases of ribosome dysfunction (ribosomopathies), such as Diamond-Blackfan anemia and 5q- syndrome, are caused by deficiencies in one or more ribosomal proteins (RPs) and are characterized by impaired erythropoiesis. The p53 pathway has been identified as a central player in the ribosomopathy disease phenotypes, but the precise mechanism by which p53 is aberrantly induced in the setting of RP deficiency has not been fully elucidated. The current hypothesis for this induction is that, in the context of RP deficiency, cellular levels of free (non-ribosome bound) RPs rise, and a subset of RPs with MDM2-binding ability interact with and inhibit the negative regulatory activity of MDM2 on p53, resulting in p53 stabilization. RPs that have been implicated in this capacity include rpL5, rpL11, rpL23a, and rpS7. This hypothesis also suggests that overabundance of these free ribosomal proteins might be sufficient to induce p53 activity, even in the absence of ribosome protein deficiency. To test this specific aspect of this hypothesis, we generated lentiviral expression vectors carrying the full-length cDNAs for rpL5, rpL11, rpL23a, and rpS7, as well as ARF (which served as a positive control, as ARF is known to induce p53 by binding and inhibiting MDM2), and we used each virus to individually infect A549 cells. Despite increased levels of transcript and protein observed for each of the cDNAs expressed, only ARF overexpression resulted in p53 stabilization and p21 induction. Similarly, when RPs were overexpressed in each pairwise combination, none of the RP pairs tested induced p53 above baseline levels. We also tested all of the overexpression constructs in primary human cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs), and p53 pathway induction was only observed with overexpression of ARF. These data suggest that overabundance of these specific ribosomal proteins alone is insufficient for p53 pathway induction via MDM2 inhibition. Disclosures: Ebert: Celgene: Consultancy; Genoptix: Consultancy.

Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.

In two separate articles published in this issue, Teisanu et al. and McQualter et al. report the use of flow cytometry and cell sorting to identify putative bronchiolar stem cells that are low in expression for the cell surface marker Sca-1 yet negative for CD34, and a mesenchymal, fibroblastic progenitor cell population from the lung that is positive for Sca-1, respectively. At first glance, these studies may seem to suggest that Sca-1 and CD34 are not markers of an epithelial stem cell population in the lung, as we previously determined in studies that identified bronchioalveolar stem cells (BASCs), and may also appear to contradict each other. However, here we point to evidence that the findings of these three studies are not mutually exclusive, and rather, that the different cell isolation and culturing protocols used in these studies have allowed for the identification of unique pulmonary cell populations. Rather than discounting previous work on BASCs, these studies reveal the existence of new methods and new cell types that will be interesting to use in future functional tests for their importance in lung biology and lung disease.