IMMU-01. TEM-GBM: AN OPEN-LABEL, PHASE I/IIA DOSE-ESCALATION STUDY EVALUATING THE SAFETY AND EFFICACY OF GENETICALLY MODIFIED TIE-2 EXPRESSING MONOCYTES TO DELIVER IFN-A WITHIN GLIOBLASTOMA TUMOR MICROENVIRONMENT

Abstract Temferon is a macrophage-based treatment relying on ex-vivo transduction of autologous HSPCs to express immune-payloads within the TME. Temferon targets the immune-modulatory molecule IFN-a, to a subset of tumor infiltrating macrophages known as Tie-2 expressing macrophages (TEMs) due to the Tie2 promoter and a post-transcriptional regulation layer represented by miRNA-126 target sequences. As of 31st May 2021, 15-patients received Temferon (D+0) with follow-up of 3 – 693 days. After conditioning neutrophil and platelet engraftment occurred at D+13 and D+13.5, respectively. Temferon-derived differentiated cells, as determined be the number of vector copy per genome, were found within 14 days post treatment and persisted albeit at lower levels up to 18-months. Very low concentrations of IFN-a in the plasma (8.7 pg/ml-D+30) and in the CSF (1.6 pg/ml-D+30) were detected, suggesting tight regulation of transgene expression. Five-deaths occurred at D+322, +340, +402, +478 and +646 due to PD, and one at D+60 due to complications following the conditioning regimen. Eight-patients had progressive disease (range: D-11 to +239) as expected for this tumor type. SAEs include GGT elevation (possibly related to Temferon) and infections, venous thromboembolism, brain abscess, hemiparesis, seizures, anemia and general physical condition deterioration, compatible with ASCT, concomitant medications and PD. Four-patients underwent 2ndsurgery. Recurrent tumors had gene-marked cells and increased expression of ISGs compared to first surgery, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased ISGs than in the progressing lesion, detected in the same patient. Tumor-associated clones expanded in the periphery. TME characterization by scRNA and TCR-sequencing is ongoing. To date, Temferon is well tolerated, with no DLTs identified. The results provide initial evidence of Temferon potential to activate the immune system of GBM patients, as predicted by preclinical studies

COMPOSITIONS AND METHODS FOR TREATING DISEASES AND DISORDERS OF THE CENTRAL NERVOUS SYSTEM

The present invention provides compositions and methods for the treatment or prevention of a neurological disease or disorder of the central nervous system (e.g., a storage disorder, lysosomal storage disorder, neurodegenerative disease, etc.) by reconstitution of brain myeloid cell and microglia upon transplantation of hematopoietic cells enriched in microglia reconstitution potential. The invention also provides compositions and methods for ablating and reconstituting microglia

Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Abstract Background Spinal cord injury (SCI) is a devastating condition mainly deriving from a traumatic damage of the spinal cord (SC). Immune cells and endogenous SC-neural stem cells (SC-NSCs) play a critical role in wound healing processes, although both are ineffective to completely restore tissue functioning. The role of SC-NSCs in SCI and, in particular, whether such cells can interplay with the immune response are poorly investigated issues, although mechanisms governing such interactions might open new avenues to develop novel therapeutic approaches. Methods We used two transgenic mouse lines to trace as well as to kill SC-NSCs in mice receiving SCI. We used Nestin CreERT2 mice to trace SC-NSCs descendants in the spinal cord of mice subjected to SCI. While mice carrying the suicide gene thymidine kinase (TK) along with the GFP reporter, under the control of the Nestin promoter regions (NestinTK mice) were used to label and selectively kill SC-NSCs. Results We found that SC-NSCs are capable to self-activate after SCI. In addition, a significant worsening of clinical and pathological features of SCI was observed in the NestinTK mice, upon selective ablation of SC-NSCs before the injury induction. Finally, mice lacking in SC-NSCs and receiving SCI displayed reduced levels of different neurotrophic factors in the SC and significantly higher number of M1-like myeloid cells. Conclusion Our data show that SC-NSCs undergo cell proliferation in response to traumatic spinal cord injury. Mice lacking SC-NSCs display overt microglia activation and exaggerate expression of pro-inflammatory cytokines. The absence of SC-NSCs impaired functional recovery as well as neuronal and oligodendrocyte cell survival. Collectively our data indicate that SC-NSCs can interact with microglia/macrophages modulating their activation/responses and that such interaction is importantly involved in mechanisms leading tissue recovery

Intracerebroventricular delivery of hematopoietic progenitors results in rapid and robust engraftment of microglia-like cells

Recent evidence indicates that hematopoietic stem and progenitor cells (HSPCs) can serve as vehicles for therapeutic molecular delivery to the brain by contributing to the turnover of resident myeloid cell populations. However, such engraftment needs to be fast and efficient to exert its therapeutic potential for diseases affecting the central nervous system. Moreover, the nature of the cells reconstituted after transplantation and whether they could comprise bona fide microglia remain to be assessed. We demonstrate that transplantation of HSPCs in the cerebral lateral ventricles provides rapid engraftment of morphologically, antigenically, and transcriptionally dependable microglia-like cells. We show that the cells comprised within the hematopoietic stem cell compartment and enriched early progenitor fractions generate this microglia-like population when injected in the brain ventricles in the absence of engraftment in the bone marrow. This delivery route has therapeutic relevance because it increases the delivery of therapeutic molecules to the brain, as shown in a humanized animal model of a prototypical lysosomal storage disease affecting the central nervous system

Lentiviral vector common integration sites in preclinical models and a clinical trial reflect a benign integration bias and not oncogenic selection

AbstractA recent clinical trial for adrenoleukodystrophy (ALD) showed the efficacy and safety of lentiviral vector (LV) gene transfer in hematopoietic stem progenitor cells. However, several common insertion sites (CIS) were found in patients' cells, suggesting that LV integrations conferred a selective advantage. We performed high-throughput LV integration site analysis on human hematopoietic stem progenitor cells engrafted in immunodeficient mice and found the same CISs reported in patients with ALD. Strikingly, most CISs in our experimental model and in patients with ALD cluster in megabase-wide chromosomal regions of high LV integration density. Conversely, cancer-triggering integrations at CISs found in tumor cells from γretroviral vector–based clinical trials and oncogene-tagging screenings in mice always target a single gene and are contained in narrow genomic intervals. These findings imply that LV CISs are produced by an integration bias toward specific genomic regions rather than by oncogenic selection

Additional file 2: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Figure S2. Sorted GFP+ cells from Nestin floxGFPflox-TK mice give rise to neurospheres. Panel A and B show the gating strategy for sorting GFP+ cells from SCs bulk cultures obtained from Nestin floxGFPflox-TK mice. WT litters (A) were used to set up the gating strategy that we used to sort GFP+ cells (B). GFP+ cells were plated at the density of 8000 cells/cm2 and daily examined for the presence of neurospheres. Small spheres were observed after 3 days (C), while spheres with diameters larger than 100 μm were easily observed after 7 days (D). Scale bar 50 μm (n = 3 independent preparations). (TIFF 9717 kb

Additional file 8: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Figure S6. Upregulation of inflammatory cues in GCV-NestinTK mice. Real-time PCR analysis of pro-inflammatory genes (A–D) in T11–T13 spinal cord tissues at different time points after the injury induction. GCV-NestinTK mice (red bars) have a increased expression of pro-inflammatory genes after injury compared with control mice (blue bars). Values indicate mean fold changes ± S.E.M (n = 3–6 for each group). Comparisons were done using the t Student test: TNFα:* p = 0.021; IL-1β:* p = 0.046; Vegfa: p = 0.008. (TIFF 7997 kb

Additional file 7: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Figure S5. macrophages/macrophages activation affects both GCV-WT and GCV-NestinTK mice. (A) Histological analysis of a SC region located 3 mm far from the injury site from a GCV-WT mice (18 days post injury). Microglia/macrophages are labeled for Iba1 and F4/80. Arrowhead indicates cells that are shown at high magnification in panel A’. Panel B shows the site of the injury in the SC of GCV-WT mouse. Arrowhead indicates cells that are shown at high magnification in panel B′. A representative section of the SC located 3 mm far from the site of the injury from a GCV-NestinTK mouse (18 days post injury) is shown in panel C. Arrowhead indicates cells that are shown at high magnification in panel C′. Panel D shows the site of the injury while the arrowhead indicates cells that are shown at high magnification in D’ (n = 3 for each group). Scale bar 50 μm (TIFF 4124 kb

Additional file 3: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Figure S3. GCV treatment ablates proliferating SC-eNSCs in NestinTK mice. Panels A and B show representative confocal images of VIM (red) and Brdu (green) in the ependymal layer of GCV-WT (A) and GCV-NestinTK (B) mice (n = 3 for each group). Mice were sacrificed at the end of the GCV treatment. Quantifications (means ± S.E.M.) are shown in panel C. Two-way ANOVA followed by Bonferroni’s multiple Comparison test has been used to analyze data. ** p = 0.011 and p = 0.045 in thoracic and lumbar segments, respectively. Scale bar 20 μm. (TIFF 2754 kb