Role Of Prior Immunity in binding to Spike of "Future" Omicron Subvariants.

BACKGROUND: Throughout the COVID-19 pandemic, virus evolution and large-scale vaccination programs have caused multiple exposures to SARS CoV-2 spike protein, resulting in complex antibody profiles. The binding of these to spike protein of "future" variants in the context of such heterogeneous exposure has not been studied. METHODS: We tested archival sera (Delta and Omicron period) stratified by anti-spike antibody (including IgG) levels for reactivity to Omicron-subvariants(BA.1, BA.2,BA.2.12.1, BA.2.75, BA.4/5 and BF.7) spike protein. Assessed antigenic distance between groups using Antigenic Cartography and performed hierarchical clustering of antibody data in a Euclidean distance framework. RESULTS: Antibody (including IgG) antibody reactivity to Wild-type (CLIA) and subvariants (ELISA) spike protein were similar between periods (p>0.05). Both 'High S' and 'Low S' of Delta and Omicron periods were closely related to "future" subvariants by Antigenic Cartography. Sera from different S groups clustered together with 'Low S' interspersed between 'High S' on hierarchical clustering, suggesting common binding sites. Further, anti-spike antibodies (including IgG) to Wild-type (S1/S2 and Trimeric S) clustered with Omicron-subvariant binding antibodies. CONCLUSIONS: Hybrid immunity caused by cumulative virus exposure in Delta or Omicron periods resulted in equivalent binding to "future" variants, which might be due to binding to conserved regions of spike protein of future variants. A prominent finding is that the 'Low S' antibody demonstrates similar binding.

SMART-lipid nanoparticles enabled mRNA vaccine elicits cross-reactive humoral responses against the omicron sub-variants.

The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has necessitated the development of broad cross-reactive vaccines. Recent findings suggest that enhanced antigen presentation could lead to cross-reactive humoral responses against the emerging variants. Toward enhancing the antigen presentation to dendritic cells (DCs), we developed a novel shikimoylated mannose receptor targeting lipid nanoparticle (SMART-LNP) system that could effectively deliver mRNAs into DCs. To improve the translation of mRNA, we developed spike domain-based trimeric S1 (TS1) mRNA with optimized codon sequence, base modification, and engineered 5' and 3' UTRs. In a mouse model, SMART-LNP-TS1 vaccine could elicit robust broad cross-reactive IgGs against Omicron sub-variants, and induced interferon-γ-producing T cells against SARS-CoV-2 virus compared with non-targeted LNP-TS1 vaccine. Further, T cells analysis revealed that SMART-LNP-TS1 vaccine induced long-lived memory T cell subsets, T helper 1 (Th1)-dominant and cytotoxic T cells immune responses against the SARS-CoV-2 virus. Importantly, SMART-LNP-TS1 vaccine produced strong Th1-predominant humoral and cellular immune responses. Overall, SMART-LNPs can be explored for precise antigenic mRNA delivery and robust immune responses. This platform technology can be explored further as a next-generation delivery system for mRNA-based immune therapies.

Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for ß-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.

Current approaches and potential challenges in the delivery of gene editing cargos into hematopoietic stem and progenitor cells.

Advancements in gene delivery and editing have expanded the applications of autologous hematopoietic stem and progenitor cells (HSPCs) for the treatment of monogenic and acquired diseases. The gene editing toolbox is growing, and the ability to achieve gene editing with mRNA or protein delivered intracellularly by vehicles, such as electroporation and nanoparticles, has highlighted the potential of gene editing in HSPCs. Ongoing phase I/II clinical trials with gene-edited HSPCs for ß-hemoglobinopathies provide hope for treating monogenic diseases. The development of safe and efficient gene editing reagents and their delivery into hard-to-transfect HSPCs have been critical drivers in the rapid translation of HSPC gene editing into clinical studies. This review article summarizes the available payloads and delivery vehicles for gene editing HSPCs and their potential impact on therapeutic applications.

Correlating the differences in the receptor binding domain of SARS-CoV-2 spike variants on their interactions with human ACE2 receptor.

Spike glycoprotein of SARS-CoV-2 variants plays a critical role in infection and transmission through its interaction with human angiotensin converting enzyme 2 (hACE2) receptors. Prior findings using molecular docking and biomolecular studies reported varied findings on the difference in the interactions among the spike variants with the hACE2 receptors. Hence, it is a prerequisite to understand these interactions in a more precise manner. To this end, firstly, we performed ELISA with trimeric spike glycoproteins of SARS-CoV-2 variants including Wuhan Hu-1(Wild), Delta, C.1.2 and Omicron. Further, to study the interactions in a more specific manner by mimicking the natural infection, we developed hACE2 receptors expressing HEK-293T cell line, evaluated their binding efficiencies and competitive binding of spike variants with D614G spike pseudotyped virus. In line with the existing findings, we observed that Omicron had higher binding efficiency compared to Delta in both ELISA and Cellular models. Intriguingly, we found that cellular models could differentiate the subtle differences between the closely related C.1.2 and Delta in their binding to hACE2 receptors. Our study using the cellular model provides a precise method to evaluate the binding interactions between spike sub-lineages to hACE2 receptors.

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of ß-hemoglobinopathies.

Reactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate ß-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to ß-globin exon-1 (ßE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets. PRR-ßE1-edited hematopoietic stem and progenitor cells (HSPCs) retained their genome integrity and their engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice producing HbF positive cells in vivo. Furthermore, PRR-ßE1 gene editing is feasible without ex vivo HSPC culture. Importantly, the editing induced therapeutically significant levels of HbF to reverse the phenotypes of both sickle cell disease and ß-thalassemia major. These findings imply that PRR-ßE1 gene editing of patient HSPCs could lead to improved therapeutic outcomes for ß-hemoglobinopathy gene therapy.

Omicron infection increases IgG binding to spike protein of predecessor variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in India in 2020-2022 was driven predominantly by Wild (Wuhan-Hu-1 and D614G), Delta, and Omicron variants. The aim of this study was to examine the effect of infections on the humoral immune response and cross-reactivity to spike proteins of Wuhan-Hu-1, Delta, C.1.2., and Omicron. Residual archival sera (N = 81) received between January 2020 and March 2022 were included. Infection status was inferred by a positive SARS-CoV-2 RT-PCR and/or serology (anti-N and anti-S antibodies) and sequencing of contemporaneous samples (N = 18) to infer lineage. We estimated the levels and cross-reactivity of infection-induced sera including Wild, Delta, Omicron as well as vaccine breakthrough infections (Delta and Omicron). We found an approximately two-fold increase in spike-specific IgG antibody binding in post-Omicron infection compared with the pre-Omicron period, whilst the change in pre- and post-Delta infections were similar. Further investigation of Omicron-specific humoral responses revealed primary Omicron infection as an inducer of cross-reactive antibodies against predecessor variants, in spite of the weaker degree of humoral response compared to Wuhan-Hu-1 and Delta infection. Intriguingly, Omicron vaccine-breakthrough infections when compared with primary infections, exhibited increased humoral responses against RBD (7.7-fold) and Trimeric S (Trimeric form of spike protein) (34.6-fold) in addition to increased binding of IgGs towards previously circulating variants (4.2 - 6.5-fold). Despite Delta breakthrough infections showing a higher level of humoral response against RBD (2.9-fold) and Trimeric S (5.7-fold) compared to primary Delta sera, a demonstrably reduced binding (36%-49%) was observed to Omicron spike protein. Omicron vaccine breakthrough infection results in increased intensity of humoral response and wider breadth of IgG binding to spike proteins of antigenically-distinct, predecessor variants.

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells.

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1Ψ-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.

Effect of Methylation of the Hydrophilic Domain of Tocopheryl Ammonium-Based Lipids on their Nucleic Acid Delivery Properties.

Lipid-enabled nucleic acid delivery has garnered tremendous attention in recent times. Tocopherol among the cationic lipids, 3b-[N-(N',N'-dimethylamino-ethane)carbamoyl]-cholesterol hydrochloride (DC-Chol) with a headgroup of dimethylammonium, and cholesterol as a hydrophobic moiety are found to be some of the most successful lipids and are being used in clinical trials. However, limited efficacy is a major limitation for their broader therapeutic application. In our prior studies, we demonstrated tocopherol to be a potential alternative hydrophobic moiety having additional antioxidant properties to develop efficient and safer liposomal formulations. Inspired by DC-Chol applications and taking cues from our own prior findings, herein, we report the design and synthesis of four alpha-tocopherol-based cationic derivatives with varying degrees of methylation, AC-Toc (no methylation), MC-Toc (monomethylation derivative), DC-Toc (dimethylation derivative), and TC-Toc (trimethylation derivative) and the evaluation of their gene delivery properties. The transfection studies showed that AC-Toc liposomes exhibited superior transfection compared to MC-Toc, DC-Toc, TC-Toc, and control DC-Chol, indicating that methylation in the hydrophilic moiety of Toc-lipids reduced their transfection properties. Cellular internalization studies in the presence of different endocytosis blockers revealed that all four tocopherol lipids were internalized through clathrin-mediated endocytosis, whereas control DC-Chol was found to be internalized through both macropinocytosis and clathrin-mediated endocytosis. These novel Toc-lipids exhibited higher antioxidant properties than DC-Chol by generating less reactive oxygen species, indicating lower cytotoxicity. Our present findings suggest that AC-Toc may be considered as an alternative to DC-Chol in liposomal transfections.

The CCR5 Gene Edited CD34+CD90+ Hematopoietic Stem Cell Population Serves as an Optimal Graft Source for HIV Gene Therapy.

Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.

Optimization of SARS-CoV-2 Pseudovirion Production in Lentivirus Backbone With a Novel Liposomal System.

Due to the fast mutating nature of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of novel therapeutics, vaccines, and evaluating the efficacies of existing one's against the mutated strains is critical for containing the virus. Pseudotyped SARS-CoV-2 viruses are proven to be instrumental in evaluating the efficiencies of therapeutics, owing to their ease in application and safety when compared to handling the live virus. However, a comprehensive protocol that includes selecting transfection reagents, validating different packaging systems for high-throughput screening of neutralizing antibodies, is still a requisite. To this end, we designed and synthesized amide linker-based cationic lipids with varying hydrophilic head groups from dimethyl (Lipo-DME) to methyl, ethylhydroxyl (Lipo-MeOH), and diethylhydroxyl (Lipo-DOH) keeping the hydrophobic tail, stearic acid, as constant. Among the liposomal formulations of these lipids, Lipo-DOH was found to be superior in delivering plasmids and demonstrated comparable transfection efficiencies with commercial standard Lipofectamine 3000. We further used Lipo-DOH for lentivirus and SARS-CoV-2 pseudovirion preparation. For comparing different lentivirus packaging systems, we optimized conditions using Addgene and BEI systems and found that the BEI lenti plasmid system was found to be efficient in making lentiviruses using Lipo-DOH. Using the optimized transfection reagent and the lentivirus system, we developed a robust protocol for the generation of SARS-CoV-2 pseudovirions and characterized their infectivity in human ACE2 expressing HEK-293T cells and neutralizing properties in IgG against spike protein of SARS-CoV-2 positive human sera from individuals recovered from COVID-19.

Skin-Permeable Nano-Lithocholic Lipidoid Efficiently Alleviates Psoriasis-like Chronic Skin Inflammations.

Long-term application of topical therapeutics for psoriasis has a plethora of side effects. Additionally, skin-permeating agents used in their formulations for deeper dermal delivery damage the skin. To address these limitations, we developed novel lithocholic acid analogues that could form lipid nanoparticles (nano-LCs) spontaneously in the aqueous milieu, permeate through the skin, penetrate the deeper dermal layers, and exert anti-inflammatory effects against psoriasis-like chronic skin inflammations. Prior findings demonstrated that lithocholic acid acts as a vitamin D receptor agonist without affecting the Ca+2 metabolism and also as an antagonist for ephrin type-A receptor 2 (EphA2). Taking cues from the previous findings, lithocholic acid derivatives with twin alkyl chains (LC6, LC8, LC10, and LC-12) were synthesized, nanoparticles (nano-LCs) were prepared, and they were evaluated for their skin permeability and anti-inflammatory properties. Among these nano-LCs, nano-LC10 demonstrated superior anti-inflammatory properties and inhibition of keratinocyte proliferation in various cell-based evaluations. Furthermore, the therapeutic efficiency of nano-LC10 was evaluated in an imiquimod-induced psoriasis-like mouse model and demonstrated comparable efficiency with the standard topical formulation, Sorvate, in reducing skin inflammations. Nano-LC10 also reduced systemic inflammation, organ toxicity, and also proinflammatory serum cytokine levels. Overall, nano-lithocholic lipidoid (nano-LC10) can be a potential novel class of therapeutics for topical application in treating psoriasis.

Influence of Hydrophobicity in the Hydrophilic Region of Cationic Lipids on Enhancing Nucleic Acid Delivery and Gene Editing.

Intracellular delivery of biomolecules using non-viral vectors critically depends on the vectors' ability to allow the escape and release of the contents from the endosomes. Prior findings demonstrated that aromatic/hydrophobic group-containing amino acids such as phenylalanine (F) and tryptophan (W) destabilize cellular membranes by forming pores in the lipid bilayer. Taking cues from these findings, we have developed four α-tocopherol-based cationic amphiphiles by varying the aromatic/hydrophobic amino acids such as glycine (G), proline (P), phenylalanine (F), and tryptophan (W) as head groups and triazole in the linker region to study their impact on endosomal escape for the enhanced transfection efficacy. The lipids tocopherol-triazole-phenylalanine (TTF) and tocopherol-triazole-tryptophan (TTW) exhibited similar potential to commercial transfecting reagents, Lipofectamine (LF) 3000 and Lipofectamine Messenger Max (LFMM), respectively, in transfecting plasmid DNA and messenger RNA in multiple cultured cell lines. The TTW liposome was also found to be effective in delivering Cas9 mRNA and demonstrated equal efficiency of gene editing AAVS1 locus compared to LFMM in CHO, Neuro-2a, and EA.HY926 cell lines. In this current investigation, it is shown that the synthesized cationic lipids with aromatic hydrophobic R group-containing amino acids are safe, economic, and actually more efficient in nucleic acid delivery and genome-editing applications. These findings can be further explored in the genome-editing approach for treating genetic disorders.

Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin.

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2.

Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.

Preferential Expansion of Human CD34+CD133+CD90+ Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy.

CD34+CD133+CD90+ hematopoietic stem cells (HSCs) are responsible for long-term multilineage hematopoiesis, and the high frequency of gene-modified HSCs is crucial for the success of hematopoietic stem and progenitor cell (HSPC) gene therapy. However, the ex vivo culture and gene manipulation steps of HSPC graft preparation significantly reduce the frequency of HSCs, thus necessitating large doses of HSPCs and reagents for the manipulation. In this study, we identified a combination of small molecules, Resveratrol, UM729, and SR1 that preferentially expands CD34+CD133+CD90+ HSCs over other subpopulations of adult HSPCs in ex vivo culture. The preferential expansion enriches the HSCs in ex vivo culture, enhances the adhesion, and results in a sixfold increase in the long-term engraftment in NSG mice. Further, the culture-enriched HSCs are more responsive to gene modification by lentiviral transduction and gene editing, increasing the frequency of gene-modified HSCs up to 10-fold in vivo. The yield of gene-modified HSCs obtained by the culture enrichment is similar to the sort-purification of HSCs and superior to Cyclosporin-H treatment. Our study addresses a critical challenge of low frequency of gene modified HSCs in HSPC graft by developing and demonstrating a facile HSPC culture condition that increases the frequency of gene-modified cells in vivo. This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

Diosgenin enhances liposome-enabled nucleic acid delivery and CRISPR/Cas9-mediated gene editing by modulating endocytic pathways.

The CRISPR/Cas9 system holds great promise in treating genetic diseases, owing to its safe and precise genome editing. However, the major challenges to implementing the technology in clinics lie in transiently limiting the expression of genome editing factors and achieving therapeutically relevant frequencies with fidelity. Recent findings revealed that non-viral vectors could be a potential alternative delivery system to overcome these limitations. In our previous research, we demonstrated that liposomal formulations with amide linker-based cationic lipids and cholesterol were found to be effective in delivering a variety of nucleic acids. In the current study, we screened steroidal sapogenins as an alternative co-lipid to cholesterol in cationic liposomal formulations and found that liposomes with diosgenin (AD, Amide lipid: Diosgenin) further improved nucleic acid delivery efficacy, in particular, delivering Cas9 pDNA and mRNA for efficient genome editing at multiple loci, including AAVS1 and HBB, when compared to amide cholesterol. Mechanistic insights into the endocytosis of lipoplexes revealed that diosgenin facilitated the lipoplexes' cholesterol-independent and clathrin-mediated endocytosis, which in turn leads to increased intracellular delivery. Our study identifies diosgenin-doped liposomes as an efficient tool to deliver CRISPR/Cas9 system.

Controlled Differentiation of Mesenchymal Stem Cells into Hyaline Cartilage in miR-140-Activated Collagen Hydrogel.

OBJECTIVE: Hypertrophic cartilage formation is a major setback in mesenchymal stem cells (MSCs)-mediated cartilage repair, and overcoming it requires optimization of differentiation. Here, we tested the miR-140 activated collagen hydrogel for the chondrogenic differentiation of MSCs and to produce hyaline cartilage. METHODS: Bone marrow MSCs isolated from 3 patients were pretreated with miR-140 and then chondrogenic differentiated. The 3-dimensional (3D) transfection potential of 5 different transfection reagents (Polyethylenimine, Lipofectamine, TransIT-X2, Amide:Cholesterol-based liposomes [AmC] and AmC pegylated with Tocofersolan [AmCTOC]) was compared and the reagent that showed higher green fluorescent protein (GFP) expression was selected. Finally, the collagen hydrogel was activated using miR-140-transfection complex and sustained delivered to MSCs during chondrogenic differentiation. After differentiation, the outcome was assessed by reverse transcription-polymerase chain reaction (RT-PCR), histology, immunohistochemistry, and compared with scrambled miRNA treated control. RESULTS: Pretreatment of MSCs with miR-140 significantly increased the expression of cartilage-specific genes (COL2A1, SOX9, and ACAN) with reduced hypertrophic chondrocyte (COL10A1) marker expression and better safranin-O staining than the control. The AmCTOC liposome showed a significant increase in 3D transfection of GFP expressing plasmid than the others. Furthermore, the knockdown of GAPDH using siRNA in HEK cells and expression of GFP mRNA in human bone marrow MSCs confirmed the 3D-transfection efficiency of AmCTOC. The sustained delivery of miR-140 using activated matrix formed a hyaline cartilage-like tissue with minimal COL10A1 expression in RT-PCR and immunohistochemistry. CONCLUSION: Our results demonstrated the therapeutic potential of miR-140-activated hydrogel for MSCs-based cartilage tissue engineering, which could also be used for endogenous stem cells-mediated cartilage repair.