A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa.

Background: Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies. Methods: We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar-free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing. Findings: iPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT-treated mice. Interpretation: DEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

Engineering human skin model innervated with itch sensory neuron-like cells differentiated from induced pluripotent stem cells.

Atopic dermatitis (AD), driven by interleukins (IL-4/IL-13), is a chronic inflammatory skin disease characterized by intensive pruritus. However, it is unclear how immune signaling and sensory response pathways cross talk with each other. We differentiated itch sensory neuron-like cells (ISNLCs) from iPSC lines. These ISNLCs displayed neural markers and action potentials and responded specifically to itch-specific stimuli. These ISNLCs expressed receptors specific for IL-4/IL-13 and were activated directly by the two cytokines. We successfully innervated these ISNLCs into full thickness human skin constructs. These innervated skin grafts can be used in clinical applications such as wound healing. Moreover, the availability of such innervated skin models will be valuable to develop drugs to treat skin diseases such as AD.

Castration-mediated IL-8 promotes myeloid infiltration and prostate cancer progression.

Unlike several other tumor types, prostate cancer rarely responds to immune checkpoint blockade (ICB). To define tumor cell intrinsic factors that contribute to prostate cancer progression and resistance to ICB, we analyzed prostate cancer epithelial cells from castration-sensitive and -resistant samples using implanted tumors, cell lines, transgenic models and human tissue. We found that castration resulted in increased expression of interleukin-8 (IL-8) and its probable murine homolog Cxcl15 in prostate epithelial cells. We showed that these chemokines drove subsequent intratumoral infiltration of tumor-promoting polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), which was largely abrogated when IL-8 signaling was blocked genetically or pharmacologically. Targeting IL-8 signaling in combination with ICB delayed the onset of castration resistance and increased the density of polyfunctional CD8 T cells in tumors. Our findings establish a novel mechanism by which castration mediates IL-8 secretion and subsequent PMN-MDSC infiltration, and highlight blockade of the IL-8/CXCR2 axis as a potential therapeutic intervention.

CRISPR/Cas9-based targeted genome editing for correction of recessive dystrophic epidermolysis bullosa using iPS cells.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in the COL7A1 gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal-epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient's quality of life. There are currently no therapies approved for the treatment of RDEB. Here, we demonstrated the correction of mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in the COL7A1 gene through homology-directed repair (HDR). We used the clustered regulatory interspaced short palindromic repeats (CRISPR) Cas9-gRNAs system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states. Three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2 mo postgrafting. Safety assessment for potential off-target Cas9 cleavage activity did not reveal any unintended nuclease activity. Our findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB.

Engineering tissue-specific blood vessels.

Vascular diversity among organs has recently become widely recognized. Several studies using mouse and human fetal tissues revealed distinct characteristics of organ-specific vasculature in molecular and functional levels. Thorough understanding of vascular heterogeneities in human adult tissues is significant for developing novel strategies for targeted drug delivery and tissue regeneration. Recent advancements in microfabrication techniques, biomaterials, and differentiation protocols allowed for incorporation of microvasculature into engineered organs. Such vascularized organ models represent physiologically relevant platforms that may offer innovative tools for dissecting the effects of the organ microenvironment on vascular development and expand our present knowledge on organ-specific human vasculature. In this article, we provide an overview of the current structural and molecular evidence on microvascular diversity, bioengineering methods used to recapitulate the microenvironmental cues, and recent vascularized three-dimensional organ models from the perspective of tissue-specific vasculature.

Brief report: embryonic growth and hatching implications of developmental 670-nm phototherapy and dioxin co-exposure.

OBJECTIVE: We assessed the effect of 670-nm light therapy on growth and hatching kinetics in chickens (Gallus gallus) exposed to dioxin. BACKGROUND DATA: Photobiomodulation has been shown to stimulate signaling pathways resulting in improved energy metabolism, antioxidant production, and cell survival. In ovo treatment with 670-nm light-emitting diode (LED) arrays improves hatching success and increases hatchling size in control chickens. Under conditions where developmental dioxin exposure is above the lethality threshold (100 ppt), phototherapy attenuates dioxin-induced early embryonic death. We hypothesized that 670-nm LED therapy would attenuate dioxin-induced developmental anomalies and increase hatching success. METHODS: Fertile chicken eggs were injected with control oil, 2, 20, or 200 ppt dioxin, or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prior to the start of incubation. Half of the eggs in each dose group were treated once per day from embryonic days 0-20 with 670-nm LED light at a fluence of 4 J/cm2. Hatchling size, organ weights, and energy parameters were compared between dose groups and LED treatment. RESULTS: LED therapy resulted in earlier pip times (small hole created 12-24 h prior to hatch), and increased hatchling size and weight in the 200 ppt dose groups. However, there appears to be an LED-oil interaction within the oil-treated controls that results in longer hatch times and decreased liver weight within the LED control dose groups in comparison to the non-LED control dose groups. CONCLUSION: Size and hatching times suggest that the hatching success and preparedness of chicks developmentally exposed to dioxin concentrations above the lethality threshold is improved by 670-nm LED treatment administered throughout the gestation period, but the relationship may be complicated by an LED-oil interaction.

How well can an idiotope peptide mimic replace its parent idiotype in a synthetic peptide vaccine?

PURPOSE: To determine whether a vaccine consisting of an idiotope peptide mimic of the third complementarity-determining region of the immunoglobulin heavy chain (CDR-H3) is an effective substitute for its parent idiotype. Such peptide vaccines could ultimately be used for targeting pathological B lymphocytes. METHODS: Hen egg lysozyme (HEL) conjugates of the Fab' fragment of monoclonal anti-fluorescein antibody 9-40 (Fab'-HEL) or a peptide mimic of the 9-40 CDR-H3 (referred to as the "B epitope" or "Bep," the conjugate is referred to as "Bep-HEL") were injected into separate cohorts of B10.A mice. Two additional control cohorts were injected with either the Bep peptide alone or a noncovalent mixture of Bep and HEL. Sera were assayed for both anti-idiotope and anti-idiotype activity by enzyme-linked immunosorbant assay (ELISA). Primary, secondary, and tertiary immune responses were examined. RESULTS: Both the Bep-HEL idiotope and the Fab-HEL idiotype immunogens elicited homologous, allogenic immune responses. No cross-reactivity was observed between anti-idiotope and anti-idiotype responses after primary immunization. With secondary immunization, 50% of mice immunized with the Bep-HEL conjugate exhibited a cross-reactive anti-idiotype response. Conversely, 100% of mice immunized with the Fab'-HEL conjugate exhibited a marginal, but statistically significant cross-reactive anti-idiotope response. Upon tertiary immunization, 100% of mice immunized with Bep-HEL exhibited a cross-reactive anti-idiotype response, and 55.6% of mice immunized with the Fab'-HEL conjugate exhibited a cross-reactive anti-idiotope response. CONCLUSIONS: Covalent coupling of a xenogenic carrier protein to an idiotype immunogen or its peptide mimic significantly enhances the intensity of homologous, allogenic anti-idiotype or anti-idiotope immune responses. Multiple immunizations are necessary to induce cross-reactivity between the peptide mimic and its parent idiotype.