Engineering Human Circulating Monocytes/Macrophages by Systemic Deliverable Gene Editing.

Delivery of plasmid DNA to transfect human primary macrophages is extremely difficult, especially for genetic engineering. Engineering macrophages is imperative for the treatment of many diseases including infectious diseases, cancer, neurological diseases, and aging. Unfortunately, plasmid does not cross the nuclear membranes of terminally differentiated macrophages to integrate the plasmid DNA (pDNA) into their genome. To address this issue, we have developed a core-shell nanoparticle (NP) using our newly created cationic lipid to deliver the anti-inflammatory cytokine IL-4 pDNA (IL-4pDNA-NPs). Human blood monocyte-derived macrophages (MDM) were effectively transfected with IL-4pDNA-NPs. IL-4pDNA-NPs were internalized in MDM within 30 minutes and delivered into the nucleus within 2 hours. Exogenous IL-4 expression was detected within 1 - 2 days and continued up to 30 days. Functional IL-4 expression led to M2 macrophage polarization in vitro and in an in vivo mouse model of inflammation. These data suggest that these NPs can protect pDNA from degradation by nucleases once inside the cell, and can transport pDNA into the nucleus to enhance gene delivery in macrophages in vitro and in vivo. In this research, we developed a new method to deliver plasmids into the nucleus of monocytes and macrophages for gene-editing. Introducing IL-4 pDNA into macrophages provides a new gene therapy solution for the treatment of various diseases.

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization.

Programmed death ligand 1 (PD-L1) plays a key role in glioblastoma multiforme (GBM) immunosuppression, vitality, proliferation, and migration, and is therefore a promising target for treating GBM. CRISPR/Cas9-mediated genomic editing can delete both cell surface and intracellular PD-L1. This systemic deliverable genomic PD-L1 deletion system can be used as an effective anti-GBM therapy by inhibiting tumor growth and migration, and overcoming immunosuppression. To target PD-L1 for CRISPR/Cas9 gene editing, we first identified two single guide RNA (sgRNA) sequences located on PD-L1 exon 3. The first sgRNA recognizes the forward strand of human PD-L1 near the beginning of exon 3 that allows editing by Cas9 at approximately base pair 82 (g82). The second sgRNA recognizes the forward strand of exon 3 that directs cutting at base pair 165 (g165). A homology-directed repair template (HDR) combined with the dual-sgRNAs was used to improve PD-L1 knockout specificity and efficiency. sgRNAs g82 and g165 were cloned into the multiplex CRISPR/Cas9 assembly system and co-transfected with the HDR template in human U87 GBM cells (g82/165 + HDR). T7E1 analysis suggests that the dual-sgRNA CRISPR/Cas9 strategy with a repair template was capable of editing the genomic level of PD-L1. This was further confirmed by examining PD-L1 protein levels by western blot and immunofluorescence assays. Western blot analysis showed that the dual-sgRNAs with the repair template caused a 64% reduction of PD-L1 protein levels in U87 cells, while immunostaining showed a significant reduction of intracellular PD-L1. PD-L1 deletion inhibited proliferation, growth, invasion and migration of U87 cells, indicating intracellular PD-L1 is necessary for tumor progression. Importantly, U87 cells treated with g82/165 + HDR polarized tumor-associated macrophages (TAM) toward an M1 phenotype, as indicated by an increase in TNF-α and a decrease in IL-4 secretions. This was further confirmed with flow cytometry that showed an increase in the M1 markers Ly6C + and CD80 +, and a decrease in the M2 marker CD206 + both in vitro and in vivo. Utilizing dual-sgRNAs and an HDR template with the CRISPR/Cas9 gene-editing system is a promising avenue for the treatment of GBM.

Cytoplasmic Trafficking of Nanoparticles Delivers Plasmid DNA for Macrophage Gene-editing.

BACKGROUND: Successful delivery of gene-editing tools using nano-carriers is dependent on the ability of nanoparticles to pass through the cellular membrane, move through the cytoplasm, and cross the nuclear envelope to enter the nucleus. It is critical that intracellular nanoparticles interact with the cytoskeletal network to move toward the nucleus, and must escape degradation pathways including lysosomal digestion. Without efficient intracellular transportation and nuclear entry, nanoparticles-based gene-editing cannot be effectively used for targeted genomic modification. OBJECTIVE: We have developed nanoparticles with a low molecular weight branched polyethylenimine lipid shell and a PLGA core that can effectively deliver plasmid DNA to macrophages for gene editing while limiting toxicity. METHODS: Core-shell nanoparticles were synthesized by a modified solvent evaporation method and were loaded with plasmid DNA. Confocal microscopy was used to visualize the internalization, intracellular distribution and cytoplasmic transportation of plasmid DNA loaded nanoparticles (pDNA-NPs) in bone marrow-derived macrophages. RESULTS: Core-shell nanoparticles had a high surface charge of +56 mV and narrow size distribution. When loaded with plasmid DNA for transfection, the nanoparticles increased in size from 150 nm to 200 nm, and the zeta potential decreased to +36 mV, indicating successful encapsulation. Further, fluorescence microscopy revealed that pDNA-NPs crossed the cell membrane and interacted with actin filaments. Intracellular tracking of pDNA-NPs showed successful separation of pDNA- NPs from lysosomes, allowing entry into the nucleus at 2 hours, with further nuclear ingress up to 5 hours. Bone marrow-derived macrophages treated with pDNA/GFP-NPs exhibited high GFP expression with low cytotoxicity. CONCLUSION: Together, this data suggests pDNA-NPs are an effective delivery system for macrophage gene-editing.

Grxcr1 Promotes Hair Bundle Development by Destabilizing the Physical Interaction between Harmonin and Sans Usher Syndrome Proteins.

Morphogenesis and mechanoelectrical transduction of the hair cell mechanoreceptor depend on the correct assembly of Usher syndrome (USH) proteins into highly organized macromolecular complexes. Defects in these proteins lead to deafness and vestibular areflexia in USH patients. Mutations in a non-USH protein, glutaredoxin domain-containing cysteine-rich 1 (GRXCR1), cause non-syndromic sensorineural deafness. To understand the deglutathionylating enzyme function of GRXCR1 in deafness, we generated two grxcr1 zebrafish mutant alleles. We found that hair bundles are thinner in homozygous grxcr1 mutants, similar to the USH1 mutants ush1c (Harmonin) and ush1ga (Sans). In vitro assays showed that glutathionylation promotes the interaction between Ush1c and Ush1ga and that Grxcr1 regulates mechanoreceptor development by preventing physical interaction between these proteins without affecting the assembly of another USH1 protein complex, the Ush1c-Cadherin23-Myosin7aa tripartite complex. By elucidating the molecular mechanism through which Grxcr1 functions, we also identify a mechanism that dynamically regulates the formation of Usher protein complexes.

4.1Ba is necessary for glutamatergic synapse formation in the sensorimotor circuit of developing zebrafish.

During the process of synapse formation, thousands of proteins assemble at prospective sites of cell-cell communication. Although many of these proteins have been identified, the roles they play in generating functional connections during development remain unknown. 4.1 scaffolding proteins have been implicated in synapse formation and maturation in vitro, but in vivo studies for some family members have suggested these proteins are not important for this role. We examined the role of family member 4.1B because it has been implicated in glutamatergic synaptogenesis, but has not been described in vivo. We identified two 4.1B genes in zebrafish, 4.1Ba and 4.1Bb, by sequence comparisons and synteny analysis. In situ hybridization shows these genes are differentially expressed, with 4.1Ba expressed primarily in the nervous system and 4.1Bb expressed in the nervous system and muscle, but not the spinal cord. We focused our studies on 4.1Ba in the spinal cord. 4.1Ba knockdown reduced the number of glutamatergic synapses at caudal primary motor neurons and caused an increase in the duration of touch-evoked coiling. These results suggest 4.1Ba is important for the formation of functional glutamatergic synapses in the developing zebrafish spinal cord.

Complexes of Usher proteins preassemble at the endoplasmic reticulum and are required for trafficking and ER homeostasis.

Usher syndrome (USH), the leading cause of hereditary combined hearing and vision loss, is characterized by sensorineural deafness and progressive retinal degeneration. Mutations in several different genes produce USH, but the proximal cause of sensory cell death remains mysterious. We adapted a proximity ligation assay to analyze associations among three of the USH proteins, Cdh23, Harmonin and Myo7aa, and the microtubule-based transporter Ift88 in zebrafish inner ear mechanosensory hair cells. We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER). Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis. ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.

Late recruitment of synapsin to nascent synapses is regulated by Cdk5.

Synapse formation is a complex process that involves the recruitment and assembly of a myriad of pre- and postsynaptic proteins. Despite being present at every synapse in the vertebrate CNS, little is known about the transport, recruitment, and stabilization of synapsin at nascent synapses during development. We examined the transport and recruitment of synapsin to nascent presynaptic terminals in vivo in the developing zebrafish spinal cord. Synapsin was transported in a transport packet independently of two other presynaptic organelles: synaptic vesicle (SV) protein transport vesicles (STVs) and Piccolo-containing active zone precursor transport vesicles (PTVs). During presynaptic assembly, recruitment of all three transport packets occurred in an ordered sequence: STVs preceded PTVs, which in turn preceded synapsin. Importantly, cyclin-dependent kinase 5 (Cdk5) specifically regulated the late recruitment of synapsin transport packets at synapses. These results point to additional layers of complexity in the established mechanisms of synaptogenesis.