Intraprocedural endothelial cell seeding of arterial stents via biotin/avidin targeting mitigates in-stent restenosis.

Impaired endothelialization of endovascular stents has been established as a major cause of in-stent restenosis and late stent thrombosis. Attempts to enhance endothelialization of inner stent surfaces by pre-seeding the stents with endothelial cells in vitro prior to implantation are compromised by cell destruction during high-pressure stent deployment. Herein, we report on the novel stent endothelialization strategy of post-deployment seeding of biotin-modified endothelial cells to avidin-functionalized stents. Acquisition of an avidin monolayer on the stent surface was achieved by consecutive treatments of bare metal stents (BMS) with polyallylamine bisphosphonate, an amine-reactive biotinylation reagent and avidin. Biotin-modified endothelial cells retain growth characteristics of normal endothelium and can express reporter transgenes. Under physiological shear conditions, a 50-fold higher number of recirculating biotinylated cells attached to the avidin-modified metal surfaces compared to bare metal counterparts. Delivery of biotinylated endothelial cells to the carotid arterial segment containing the implanted avidin-modified stent in rats results in immediate cell binding to the stent struts and is associated with a 30% reduction of in-stent restenosis in comparison with BMS.

Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1.

In-stent restenosis (ISR) complicates revascularization in the coronary and peripheral arteries. Apolipoprotein A1 (apoA1), the principal protein component of HDL possesses inherent anti-atherosclerotic and anti-restenotic properties. These beneficial traits are lost when wild type apoA1(WT) is subjected to oxidative modifications. We investigated whether local delivery of adeno-associated viral (AAV) vectors expressing oxidation-resistant apoA1(4WF) preserves apoA1 functionality. The efflux of 3H-cholesterol from macrophages to the media conditioned by endogenously produced apoA1(4WF) was 2.1-fold higher than for apoA1(WT) conditioned media in the presence of hypochlorous acid emulating conditions of oxidative stress. The proliferation of apoA1(WT)- and apoA1(4FW)-transduced rat aortic smooth muscle cells (SMC) was inhibited by 66% ± 10% and 65% ± 11%, respectively, in comparison with non-transduced SMC (p < 0.001). Conversely, the proliferation of apoA1(4FW)-transduced, but not apoA1(WT)-transduced rat blood outgrowth endothelial cells (BOEC) was increased 41% ± 5% (p < 0.001). Both apoA1 transduction conditions similarly inhibited basal and TNFα-induced reactive oxygen species in rat aortic endothelial cells (RAEC) and resulted in the reduced rat monocyte attachment to the TNFα-activated endothelium. AAV2-eGFP vectors immobilized reversibly on stainless steel mesh surfaces through the protein G/anti-AAV2 antibody coupling, efficiently transduced cells in culture modeling stent-based delivery. In vivo studies in normal pigs, deploying AAV2 gene delivery stents (GDS) preloaded with AAV2-eGFP in the coronary arteries demonstrated transduction of the stented arteries. However, implantation of GDS formulated with AAV2-apoA1(4WF) failed to prevent in-stent restenosis in the atherosclerotic vasculature of hypercholesterolemic diabetic pigs. It is concluded that stent delivery of AAV2-4WF while feasible, is not effective for mitigation of restenosis in the presence of severe atherosclerotic disease.

Ionizable lipid nanoparticles for in utero mRNA delivery.

Clinical advances enable the prenatal diagnosis of genetic diseases that are candidates for gene and enzyme therapies such as messenger RNA (mRNA)-mediated protein replacement. Prenatal mRNA therapies can treat disease before the onset of irreversible pathology with high therapeutic efficacy and safety due to the small fetal size, immature immune system, and abundance of progenitor cells. However, the development of nonviral platforms for prenatal delivery is nascent. We developed a library of ionizable lipid nanoparticles (LNPs) for in utero mRNA delivery to mouse fetuses. We screened LNPs for luciferase mRNA delivery and identified formulations that accumulate within fetal livers, lungs, and intestines with higher efficiency and safety compared to benchmark delivery systems, DLin-MC3-DMA and jetPEI. We demonstrate that LNPs can deliver mRNAs to induce hepatic production of therapeutic secreted proteins. These LNPs may provide a platform for in utero mRNA delivery for protein replacement and gene editing.

In utero CRISPR-mediated therapeutic editing of metabolic genes.

In utero gene editing has the potential to prenatally treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. We assessed the viral vector-mediated delivery of CRISPR-Cas9 or base editor 3 in utero, seeking therapeutic modification of Pcsk9 or Hpd in wild-type mice or the murine model of hereditary tyrosinemia type 1, respectively. We observed long-term postnatal persistence of edited cells in both models, with reduction of plasma PCSK9 and cholesterol levels following in utero Pcsk9 targeting and rescue of the lethal phenotype of hereditary tyrosinemia type 1 following in utero Hpd targeting. The results of this proof-of-concept work demonstrate the possibility of efficiently performing gene editing before birth, pointing to a potential new therapeutic approach for selected congenital genetic disorders.

Tyrosine 870 of TLR9 is critical for receptor maturation rather than phosphorylation-dependent ligand-induced signaling.

Toll like receptors (TLRs) share a conserved structure comprising the N-terminal ectodomain, a transmembrane segment and a C-terminal cytoplasmic Toll/IL-1 receptor (TIR) domain. Proper assembly of the TIR domain is crucial for signal transduction; however, the contribution of individual motifs within the TIR domain to TLR trafficking and signaling remains unclear. We targeted a highly conserved tyrosine (Y870) located in the box 1 region of the TIR domain of most TLRs, including TLR9, previously described to be a critical site of phosphorylation in TLR4. We reconstituted bone marrow-derived dendritic cells (BMDC) from Tlr9-/- mice WT TLR9 or Y870F or Y870A mutants. Despite normal interactions with the luminal chaperones GRP94 and UNC93B1, Y870F conferred only partial responsiveness to CpG, and Y870A had no activity and functioned as a dominant negative inhibitor when coexpressed with endogenous TLR9. This loss of function correlated with reduction or absence, respectively, of the 80 kDa mature form of TLR9. In Y870F-expressing cells, CpG-dependent signaling correlated directly with levels of the mature form, suggesting that signaling did not require tyrosine phosphorylation but rather that the Y870F mutation conferred reduced receptor levels due to defective processing or trafficking. Microscopy revealed targeting of the mutant protein to an autophagolysosome-like structure for likely degradation. Collectively we postulate that the conserved Y870 in the TIR domain does not participate in phosphorylation-induced signaling downstream of ligand recognition, but rather is crucial for proper TIR assembly and ER egress, resulting in maturation-specific stabilization of TLR9 within endolysosomes and subsequent pro-inflammatory signaling.

"Marker of Self" CD47 on lentiviral vectors decreases macrophage-mediated clearance and increases delivery to SIRPA-expressing lung carcinoma tumors.

Lentiviruses infect many cell types and are now widely used for gene delivery in vitro, but in vivo uptake of these foreign vectors by macrophages is a limitation. Lentivectors are produced here from packaging cells that overexpress "Marker of Self" CD47, which inhibits macrophage uptake of cells when prophagocytic factors are also displayed. Single particle analyses show "hCD47-Lenti" display properly oriented human-CD47 for interactions with the macrophage's inhibitory receptor SIRPA. Macrophages derived from human and NOD/SCID/Il2rg-/- (NSG) mice show a SIRPA-dependent decrease in transduction, i.e., transgene expression, by hCD47-Lenti compared to control Lenti. Consistent with known "Self" signaling pathways, macrophage transduction by control Lenti is decreased by drug inhibition of Myosin-II to the same levels as hCD47-Lenti. In contrast, human lung carcinoma cells express SIRPA and use it to enhance transduction by hCD47-Lenti- as illustrated by more efficient gene deletion using CRISPR/Cas9. Intravenous injection of hCD47-Lenti into NSG mice shows hCD47 prolongs circulation, unless a blocking anti-SIRPA is preinjected. In vivo transduction of spleen and liver macrophages also decreases for hCD47-Lenti while transduction of lung carcinoma xenografts increases. hCD47 could be useful when macrophage uptake is limiting on other viral vectors that are emerging in cancer treatments (e.g., Measles glycoprotein-pseudotyped lentivectors) and also in targeting various SIRPA-expressing tumors such as glioblastomas.

Life-Long Transgene Expression in Skeletal Muscle Without Transduction of Satellite Cells Following Embryonic Myogenic Progenitor Transduction by Lentivirus Administered in Utero.

Embryologic events in mammalian myogenesis remain to be fully defined. Recent evidence supports the presence of a common progenitor arising in the dermomyotome that gives rise to both embryologic and adult muscle and postnatal myogenic stem cells (satellite cells). In this study, we utilize the technique of early intra-amniotic gene transfer to target nascent muscle progenitors as they traverse the primitive streak before formation of the dermomyotome. This technique robustly transduced both epaxial and hypaxial muscle groups. Marker gene expression is observed in up to 100% muscle fibers in the lower extremities and is sustained for the lifetime of the mouse. We next analyzed transduced muscle for satellite cell transduction using highly sensitive methodology. Surprisingly, despite high levels of sustained transgene expression in muscle fibers, satellite cells lacked the marker transgene. Our data suggest that dermatomyotome is a heterogeneous structure and that not all myogenic progenitors of dermatomyotome give rise to satellite cells.

In utero lung gene transfer using adeno-associated viral and lentiviral vectors in mice.

Virus-mediated gene transfer to the fetal lung epithelium holds considerable promise for the therapeutic management of prenatally diagnosed, potentially life-threatening inherited lung diseases. In this study we hypothesized that efficient and life-long lung transduction can be achieved by in utero gene therapy, using viral vectors. To facilitate diffuse entry into the lung, viral vector was injected into the amniotic sac of C57BL/6 mice on embryonic day 16 (term, ∼ 20 days) in a volume of 10 µl. Vectors investigated included those based on adeno-associated virus (AAV) (serotypes 5, 6.2, 9, rh.64R1) and vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1-based lentivirus (LV). All vectors expressed green fluorescent protein (GFP) under the transcriptional control of various promoters including chicken ß-actin (CB) or cytomegalovirus (CMV) for AAV and CMV or MND (myeloproliferative sarcoma virus enhancer, negative control region deleted) for LV. Pulmonary GFP gene expression was detected by fluorescence stereoscopic microscopy and immunohistochemistry for up to 9 months after birth. At equivalent vector doses (mean, 12 × 10(10) genome copies per fetus) three AAV vectors resulted in long-term (up to 9 months) pulmonary epithelium transduction. AAV2/6.2 transduced predominantly cells of the conducting airway epithelium, although transduction decreased 2 months after vector delivery. AAV2/9-transduced cells of the alveolar epithelium with a type 1 pneumocyte phenotype for up to 6 months. Although minimal levels of GFP expression were observed with AAV2/5 up to 9 months, the transduced cells immunostained positive for F480 and were retrievable by bronchoalveolar lavage, confirming an alveolar macrophage phenotype. No GFP expression was observed in lung epithelial cells after AAV2/rh.64R1 and VSV-G-LV vector-mediated gene transfer. We conclude that these experiments demonstrate that prenatal lung gene transfer with AAV vectors engineered to target pulmonary epithelial cells may provide sustained long-term levels of transgene expression, supporting the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.

TLR ligands up-regulate Trex1 expression in murine conventional dendritic cells through type I Interferon and NF-κB-dependent signaling pathways.

Mutations in the Trex1 are associated with a spectrum of type I IFN-dependent autoimmune diseases. Trex1 plays an essential role in preventing accumulation of excessive cytoplasmic DNA, avoiding cell-intrinsic innate DNA sensor activation and suppressing activation of type I IFN-stimulated and -independent antiviral genes. Trex1 also helps HIV to escape cytoplasmic detection by DNA sensors. However, regulation of Trex1 in innate immune cells remains elusive. We report that murine cDCs have high constitutive expression of Trex1 in vitro and in vivo in the spleen. In resting bone marrow-derived cDCs, type I IFNs up-regulate Trex1 expression via the IFNAR-mediated signaling pathway (STAT1- and STAT2-dependent). DC activation induced by TLR3, -4, -7, and -9 ligands also augments Trex1 expression through autocrine IFN-ß production and triggering of the IFN signaling pathway, whereas TLR4 ligand LPS also stimulates an early expression of Trex1 through IFN-independent NF-κB-dependent signaling pathway. Furthermore, retroviral infection also induces Trex1 up-regulation in cDCs, as we found that a gene therapy HIV-1-based lentiviral vector induces significant Trex1 expression, suggesting that Trex1 may affect local and systemic administration of gene-therapy vehicles. Our data indicate that Trex1 is induced in cDCs during activation upon IFN and TLR stimulation through the canonical IFN signaling pathway and suggest that Trex1 may play a role in DC activation during infection and autoimmunity. Finally, these results suggest that HIV-like viruses may up-regulate Trex1 to increase their ability to escape immunosurveillance.

IL-4 suppresses the responses to TLR7 and TLR9 stimulation and increases the permissiveness to retroviral infection of murine conventional dendritic cells.

Th2-inducing pathological conditions such as parasitic diseases increase susceptibility to viral infections through yet unclear mechanisms. We have previously reported that IL-4, a pivotal Th2 cytokine, suppresses the response of murine bone-marrow-derived conventional dendritic cells (cDCs) and splenic DCs to Type I interferons (IFNs). Here, we analyzed cDC responses to TLR7 and TLR9 ligands, R848 and CpGs, respectively. We found that IL-4 suppressed the gene expression of IFNß and IFN-responsive genes (IRGs) upon TLR7 and TLR9 stimulation. IL-4 also inhibited IFN-dependent MHC Class I expression and amplification of IFN signaling pathways triggered upon TLR stimulation, as indicated by the suppression of IRF7 and STAT2. Moreover, IL-4 suppressed TLR7- and TLR9-induced cDC production of pro-inflammatory cytokines such as TNFα, IL-12p70 and IL-6 by inhibiting IFN-dependent and NFκB-dependent responses. IL-4 similarly suppressed TLR responses in splenic DCs. IL-4 inhibition of IRGs and pro-inflammatory cytokine production upon TLR7 and TLR9 stimulation was STAT6-dependent, since DCs from STAT6-KO mice were resistant to the IL-4 suppression. Analysis of SOCS molecules (SOCS1, -2 and -3) showed that IL-4 induces SOCS1 and SOCS2 in a STAT6 dependent manner and suggest that IL-4 suppression could be mediated by SOCS molecules, in particular SOCS2. IL-4 also decreased the IFN response and increased permissiveness to viral infection of cDCs exposed to a HIV-based lentivirus. Our results indicate that IL-4 modulates and counteracts pro-inflammatory stimulation induced by TLR7 and TLR9 and it may negatively affect responses against viruses and intracellular parasites.

Intra-amniotic transient transduction of the periderm with a viral vector encoding TGFß3 prevents cleft palate in Tgfß3(-/-) mouse embryos.

Cleft palate is a developmental defect resulting from the failure of embryonic palatal shelves to fuse with each other at a critical time. Immediately before and during palatal fusion (E13-E15 in mice), transforming growth factor ß3 (TGFß3) is expressed in the palatal shelf medial edge epithelium (MEE) and plays a pivotal role in palatal fusion. Using Tgfß3(-/-) mice, which display complete penetrance of the cleft palate phenotype, we tested the hypothesis that intra-amniotic gene transfer could be used to prevent cleft palate formation by restoring palatal midline epithelial function. An adenoviral vector encoding Tgfß3 was microinjected into the amniotic sacs of mouse embryos at successive developmental stages. Transduced Tgfß3(-/-) fetuses showed efficient recovery of palatal fusion with mesenchymal confluence following injection at E12.5 (100%), E13.5 (100%), E14.5 (82%), and E15.5 (75%). Viral vectors injected into the amniotic sac transduced the most superficial and transient peridermal cell layer but not underlying basal epithelial cells. TGFß3 transduction of the peridermdal cell layer was sufficient to induce adhesion, fusion, and disappearance of the palatal shelf MEE in a cell nonautonomous manner. We propose that intra-amniotic gene transfer approaches have therapeutic potential to prevent cleft palate in utero, especially those resulting from palatal midline epithelial dysfunction.

Robust in vivo transduction of nervous system and neural stem cells by early gestational intra amniotic gene transfer using lentiviral vector.

Presently, in vivo methods to efficiently and broadly transduce all major cell types throughout both the central (CNS) and peripheral adult nervous system (PNS) are lacking. In this study, we hypothesized that during early fetal development neural cell populations, including neural stem cells (NSCs), may be accessible for gene transfer via the open neural groove. To test this hypothesis, we injected lentiviral vectors encoding a green fluorescent protein (GFP) marker gene into the murine amniotic cavity at embryonic day 8. This method (i) efficiently and stably transduced the entire nervous system for at least 80% of the lifespan of the mice, (ii) transduced all major neural cell types, and (iii) transduced adult NSCs of the subventricular zone (SVZ) and subgranular zones (SGZs). This simple approach has broad applications for the study of gene function in nervous system development and adult NSCs and may have future clinical applications for treatment of genetic disorders of the nervous system.

Placental gene transfer: transgene screening in mice for trophic effects on the placenta.

OBJECTIVE: We hypothesized that gene transfer of select growth factors to the placenta may enhance placental and fetal growth. Thus, we examined the effect of 8 growth factor transgenes on murine placenta. STUDY DESIGN: Adenoviral-mediated site-specific intraplacental gene transfer of 8 different growth factor transgenes at embryonic day (e) 14 was performed. Transgenes included angiopoietin-1, angiopoietin-2 (Ang-2), basic fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor-1 (IGF-1), placenta growth hormone, platelet-derived growth factor-B (PDGF-B), and vascular endothelial growth factor(121). Fetuses and placentas were harvested at e17 and assessed for survival, gene transfer efficiency, placenta area, and fetal and placental weights. RESULTS: Efficient gene transfer to the placenta was detected with minimal dissemination to the fetus. Overexpression of IGF-1, PDGF-B, and Ang-2 resulted in an increase in placenta cross-sectional area. Only Ang-2 gene transfer resulted in increased fetal weight, and only Ang-2 and basic fibroblast growth factor resulted in a change in placental weight. CONCLUSION: Site-specific placental gene transfer results in efficient gene transfer with minimal dissemination to the fetus. Adenoviral-mediated IGF-1, adenoviral-mediated PDGF-B, and adenoviral-mediated Ang-2 significantly increase placenta growth.

Maternal alloantibodies induce a postnatal immune response that limits engraftment following in utero hematopoietic cell transplantation in mice.

The lack of fetal immune responses to foreign antigens, i.e., fetal immunologic tolerance, is the most compelling rationale for prenatal stem cell and gene therapy. However, the frequency of engraftment following in utero hematopoietic cell transplantation (IUHCT) in the murine model is reduced in allogeneic, compared with congenic, recipients. This observation supports the existence of an immune barrier to fetal transplantation and challenges the classic assumptions of fetal tolerance. Here, we present evidence that supports the presence of an adaptive immune response in murine recipients of IUHCT that failed to maintain engraftment. However, when IUHCT recipients were fostered by surrogate mothers, they all maintained long-term chimerism. Furthermore, we have demonstrated that the cells responsible for rejection of the graft were recipient in origin. Our observations suggest a mechanism by which IUHCT-dependent sensitization of the maternal immune system and the subsequent transmission of maternal alloantibodies to pups through breast milk induces a postnatal adaptive immune response in the recipient, which, in turn, results in the ablation of engraftment after IUHCT. Finally, we showed that non-fostered pups that maintained their chimerism had higher levels of Tregs as well as a more suppressive Treg phenotype than their non-chimeric, non-fostered siblings. This study resolves the apparent contradiction of induction of an adaptive immune response in the pre-immune fetus and confirms the potential of actively acquired tolerance to facilitate prenatal therapeutic applications.

Correction of murine ADAMTS13 deficiency by hematopoietic progenitor cell-mediated gene therapy.

ADAMTS13, a metalloprotease primarily synthesized in liver and endothelial cells, cleaves von Willebrand factor (VWF) at the central A2 domain, thereby reducing the sizes of circulating VWF multimers. Genetic or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). To date, plasma infusion or exchange is the only proven effective therapy for TTP. In search for a better therapy, an autologous transplantation of hematopoietic progenitor cells transduced ex vivo with a self-inactivating lentiviral vector encoding a full-length murine Adamts13 and an enhanced green fluorescent protein (GFP) reporter gene was performed in Adamts13(-/-) mice after irradiation. All recipient mice showed detectable ADAMTS13 antigen and proteolytic activity in plasma despite only low levels of bone marrow chimerism. The levels of plasma ADAMTS13 were sufficient to eliminate the ultralarge VWF multimers and offered systemic protection against ferric chloride-induced arterial thrombosis. The data suggest that hematopoietic progenitor cells can be genetically modified ex vivo and transplanted in an autologous model to provide adequate levels of functional ADAMTS13 metalloprotease. This success may provide the basis for development of a novel therapeutic strategy to cure hereditary TTP in humans.

Correction of ADAMTS13 deficiency by in utero gene transfer of lentiviral vector encoding ADAMTS13 genes.

Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (approximately 5-70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor (vWF) compared to the Adamts13(-/-) mice expressing enhanced green fluorescent protein (eGFP). Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13(-/-) expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13(-/-) mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.

Apoptotic cell-mediated immunoregulation of dendritic cells does not require iC3b opsonization.

A number of recent studies show that activation of CR3 on dendritic cells (DCs) suppresses TLR-induced TNF-alpha and IL-12 production and inhibits effective Ag presentation. Although the proposed physiologic role for these phenomena is immune suppression due to recognition of iC3b opsonized apoptotic cells by CR3, all of the aforementioned investigations used artificial means of activating CR3. We investigated whether iC3b opsonized apoptotic cells could induce the same changes reported with artificial ligands such as mAbs or iC3b-opsonized RBC. We explored the kinetics of iC3b opsonization in two models of murine cell apoptosis, gamma-irradiated thymocytes and cytokine deprivation of the IL-3 dependent cell line BaF3. Using a relatively homogenous population of early apoptotic cells (IL-3 deprived BaF3 cells), we show that iC3b opsonized apoptotic cells engage CR3, but this interaction is dispensable in mediating the anti-inflammatory effects of apoptotic cells. TLR-induced TNF-alpha and IL-12 production by bone marrow-derived DCs occurs heterogeneously, with apoptotic cells inhibiting only certain populations depending on the TLR agonist. In contrast, although apoptotic cells induced homogeneous IL-10 production by DCs, IL-10 was not necessary for the inhibition of TNF-alpha and IL-12. Furthermore, because the ability of iC3b opsonization to enhance phagocytosis of apoptotic cells has been controversial, we report that iC3b opsonization does not significantly affect apoptotic cell ingestion by DCs. We conclude that the apoptotic cell receptor system on DCs is sufficiently redundant such that the absence of CR3 engagement does not significantly affect the normal anti-inflammatory processing of apoptotic cells.

Cystic adenomatoid malformations are induced by localized FGF10 overexpression in fetal rat lung.

Fibroblast growth factor-10 (FGF10) is a mesenchymal growth factor, involved in epithelial and mesenchymal interactions during lung branching morphogenesis. In the present work, FGF10 overexpression was transiently induced in a temporally and spatially restricted manner, during the pseudoglandular or canalicular stages of rat lung development, by trans-uterine ultrasound-guided intraparenchymal microinjections of adenoviral vector encoding the rfgf10 transgene. The morphologic and histologic classification of the resulting malformations were dependent upon developmental stage and location. Overexpression of FGF10 restricted to the proximal tracheobronchial tree during the pseudoglandular phase resulted in large cysts lined by tall columnar epithelium composed primarily of Clara cells with a paucity of Type II pneumocytes, resembling bronchiolar type epithelium. In contrast, FGF10 overexpression in the distal lung parenchyma during the canalicular phase resulted in small cysts lined by cuboidal epithelial cells composed of primarily Type II pneumocytes resembling acinar epithelial differentiation. The cystic malformations induced by FGF10 overexpression appear to closely recapitulate the morphology and histology of the spectrum of human congenital cystic adenomatoid malformation (CCAM). These findings support a role for FGF10 in the induction of human CCAM and provide further mechanistic insight into the role of FGF10 in normal and abnormal lung development.

IL-10 overexpression decreases inflammatory mediators and promotes regenerative healing in an adult model of scar formation.

Adult wound healing is characterized by an exuberant inflammatory response and scar formation. In contrast, scarless fetal wound healing has diminished inflammation, a lack of fibroplasia, and restoration of normal architecture. We have previously shown that fetal wounds produce less inflammatory cytokines, and the absence of IL-10, an anti-inflammatory cytokine, results in fetal scar formation. We hypothesized that increased IL-10 would decrease inflammation and create an environment conducive for regenerative healing in the adult. To test this hypothesis, a lentiviral vector expressing IL-10 and green fluorescent protein (GFP) (Lenti-IL-10) or GFP alone (Lenti-GFP) was injected at the wound site 48 hours before wounding. We found that both Lenti-IL-10 and Lenti-GFP were expressed in the wounds at 1 and 3 days post wounding. At 3 days, Lenti-IL-10-treated wounds demonstrated decreased inflammation and decreased quantities of all proinflammatory mediators analyzed with statistically different levels of IL-6, monocyte chemoattractant protein-1, and heat-shock protein 47. At 3 weeks, Lenti-GFP wounds demonstrated scar formation. In contrast, wounds injected with Lenti-IL-10 demonstrated decreased inflammation, a lack of abnormal collagen deposition, and restoration of normal dermal architecture. We conclude that lentivirus-mediated overexpression of IL-10 decreases the inflammatory response to injury, creating an environment conducive for regenerative adult wound healing.