Chemokine-enhanced DNA vaccination in cancer immunotherapy.

We have demonstrated that priming of intratumoral and intradermal vaccination sites with chemokines enhances cytotoxic immune response against established neoplasms. Additional insights into the molecular mechanisms that underlie these findings and the optimization of such an approach may lead to the development of cost-effective and generic immunotherapeutic regimens against cancer

Pro-Inflammatory Chemokines and Cytokines Dominate the Blister Fluid Molecular Signature in Patients with Epidermolysis Bullosa and Affect Leukocyte and Stem Cell Migration.

Hereditary epidermolysis bullosa (EB) is associated with skin blistering and the development of chronic nonhealing wounds. Although clinical studies have shown that cell-based therapies improve wound healing, the recruitment of therapeutic cells to blistering skin and to more advanced skin lesions remains a challenge. Here, we analyzed cytokines and chemokines in blister fluids of patients affected by dystrophic, junctional, and simplex EB. Our analysis revealed high levels of CXCR1, CXCR2, CCR2, and CCR4 ligands, particularly dominant in dystrophic and junctional EB. In vitro migration assays demonstrated the preferential recruitment of CCR4+ lymphocytes and CXCR1+, CXCR2+, and CCR2+ myeloid cells toward EB-derived blister fluids. Immunophenotyping of skin-infiltrating leukocytes confirmed substantial infiltration of EB-affected skin with resting (CD45RA+) and activated (CD45RO+) T cells and CXCR2+ CD11b+ cells, many of which were identified as CD16b+ neutrophils. Our studies also showed that abundance of CXCR2 ligand in blister fluids also creates a favorable milieu for the recruitment of the CXCR2+ stem cells, as validated by in vitro and in-matrix migration assays. Collectively, this study identified several chemotactic pathways that control the recruitment of leukocytes to the EB-associated skin lesions. These chemotactic axes could be explored for the refinement of the cutaneous homing of the therapeutic stem cells. © 2017 The Author

Ladarixin, a dual CXCR1/2 inhibitor, attenuates experimental melanomas harboring different molecular defects by affecting malignant cells and tumor microenvironment.

CXCR1 and CXCR2 chemokine receptors and their ligands (CXCL1/2/3/7/8) play an important role in tumor progression. Tested to date CXCR1/2 antagonists and chemokine-targeted antibodies were reported to affect malignant cells in vitro and in animal models. Yet, redundancy of chemotactic signals and toxicity hinder further clinical development of these approaches. In this pre-clinical study we investigated the capacity of a novel small molecule dual CXCR1/2 inhibitor, Ladarixin (LDX), to attenuate progression of experimental human melanomas. Our data showed that LDX-mediated inhibition of CXCR1/2 abrogated motility and induced apoptosis in cultured cutaneous and uveal melanoma cells and xenografts independently of the molecular defects associated with the malignant phenotype. These effects were mediated by the inhibition of AKT and NF-kB signaling pathways. Moreover, systemic treatment of melanoma-bearing mice with LDX also polarized intratumoral macrophages to M1 phenotype, abrogated intratumoral de novo angiogenesis and inhibited melanoma self-renewal. Collectively, these studies outlined the pre-requisites of the successful CXCR1/2 inhibition on malignant cells and demonstrated multifactorial effects of Ladarixin on cutaneous and uveal melanomas, suggesting therapeutic utility of LDX in treatment of various melanoma types

Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion

Genome engineering using single-stranded oligonucleotides is an efficient method for generating small chromosomal and episomal modifications in a variety of host organisms. The efficiency of this allelic replacement strategy is highly dependent on avoidance of the endogenous mismatch repair (MMR) machinery. However, global MMR inactivation generally results in significant accumulation of undesired background mutations. Here, we present a novel strategy using oligos containing chemically modified bases (2′-Fluoro-Uridine, 5-Methyl-deoxyCytidine, 2,6-Diaminopurine or Iso-deoxyGuanosine) in place of the standard T, C, A or G to avoid mismatch detection and repair, which we tested in Escherichia coli. This strategy increases transient allelic-replacement efficiencies by up to 20-fold, while maintaining a 100-fold lower background mutation level. We further show that the mismatched bases between the full length oligo and the chromosome are often not incorporated at the target site, probably due to nuclease activity at the 5′ and 3′ termini of the oligo. These results further elucidate the mechanism of oligo-mediated allelic replacement (OMAR) and enable improved methodologies for efficient, large-scale engineering of genomes.Synthetic Biology Engineering Research CenterNational Science Foundation (U.S.) (Grant #SA5283-11210)United States. Dept. of Energy (Genomes to Life Center) (Grant #DE-FG02-03ER6344)Wyss Institute for Biologically Inspired Engineerin

Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells

Oligonucleotide-mediated gene targeting is emerging as a powerful tool for the introduction of subtle gene modifications in mouse embryonic stem (ES) cells and the generation of mutant mice. However, its efficacy is strongly suppressed by DNA mismatch repair (MMR). Here we report a simple and rapid procedure for the generation of mouse mutants using transient down regulation of the central MMR protein MSH2 by RNA interference. We demonstrate that under this condition, unmodified single-stranded DNA oligonucleotides can be used to substitute single or several nucleotides. In particular, simultaneous substitution of four adjacent nucleotides was highly efficient, providing the opportunity to substitute virtually any given codon. We have used this method to create a codon substitution (N750F) in the Rb gene of mouse ES cells and show that the oligonucleotide-modified Rb allele can be transmitted through the germ line of mice

Congenital muscular dystrophy-associated inflammatory chemokines provide axes for effective recruitment of therapeutic adult stem cell into muscles

Background: Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness. The two most prevalent forms of CMD, collagen VI-related myopathies (COL6RM) and laminin α2 deficient CMD type 1A (MDC1A), are both caused by deficiency or dysfunction of extracellular matrix proteins. Previously, we showed that an intramuscular transplantation of human adipose-derived stem cells (ADSC) into the muscle of the Col6a1-/- mice results in efficient stem cell engraftment, migration, long-term survival, and continuous production of the collagen VI protein, suggesting the feasibility of the systemic cellular therapy for COL6RM. In order for this therapeutic approach to work however, stem cells must be efficiently targeted to the entire body musculature. Thus, the main goal of this study is to test whether muscle homing of systemically transplanted ADSC can be enhanced by employing muscle-specific chemotactic signals originating from CMD-affected muscle tissue. Methods: Proteomic screens of chemotactic molecules were conducted in the skeletal muscles of COL6RM- and MDC1A-affected patients and CMD mouse models to define the inflammatory and immune activities, thus, providing potential markers of disease activity or treatment effect. Also using a pre-clinical animal model, recapitulating mild Ullrich congenital muscular dystrophy (UCMD), the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations. Results: Comprehensive proteomic screens evaluating relevant human and mouse skeletal muscle biopsies offered chemotactic axes to enhance directional migration of systemically transplanted cells into CMD-affected muscles, including CCL5-CCR1/3/5, CCL2-CCR2, CXCL1/2-CXCR1,2, and CXCL7-CXCR2. Also, the specific populations of ADSC selected with an affinity for the chemokines being released by damaged muscle showed efficient migration to injured site and presented their therapeutic effect. Conclusions: Collectively, identified molecules provided insight into the mechanisms governing directional migration and intramuscular trafficking of systemically infused stem cells, thus, permitting broad and effective application of the therapeutic adult stem cells for CMD treatment

Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides

Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested

RecA-mediated strand invasion of DNA by oligonucleotides substituted with 2-aminoadenine and 2-thiothymine

Sequence-specific recognition of DNA is a critical step in gene targeting. Here we describe unique oligonucleotide (ON) hybrids that can stably pair to both strands of a linear DNA target in a RecA-dependent reaction with ATP or ATPγS. One strand of the hybrids is a 30-mer DNA ON that contains a 15-nt-long A/T-rich central core. The core sequence, which is substituted with 2-aminoadenine and 2-thiothymine, is weakly hybridized to complementary locked nucleic acid or 2′-OMe RNA ONs that are also substituted with the same base analogs. Robust targeting reactions took place in the presence of ATPγS and generated metastable double D-loop joints. Since the hybrids had pseudocomplementary character, the component ONs hybridized less strongly to each other than to complementary target DNA sequences composed of regular bases. This difference in pairing strength promoted the formation of joints capable of accommodating a single mismatch. If similar joints can form in vivo, virtually any A/T-rich site in genomic DNA could be selectively targeted. By designing the constructs so that the DNA ON is mismatched to its complementary sequence in DNA, joint formation might allow the ON to function as a template for targeted point mutation and gene correction