Novel Expansions and Trends in Gene Diagnostics and Gene Therapy

A special issue of Bioengineering (ISSN 2306-5354). This special issue belongs to the section "Biomedical Engineering and Biomaterials".

Deadline for manuscript submissions: closed (30 June 2023) | Viewed by 22446

Special Issue Editors

1. 2nd Department of Pediatrics, Semmelweis University, Budapest, Hungary
2. For Human Genome Foundation, Budapest, Hungary
Interests: molecular genetics; CRISPR-Cas9; nanopore sequencing; regulatory T-cells; single-cell multiomics; flow cytometry; clinical genetics
2nd Department of Pediatrics, Semmelweis University, Budapest, Hungary
Interests: molecular genetic studies; clinical genetics

Special Issue Information

Dear Colleagues, 

Technological advances in sequencing technologies and the ongoing deciphering studies of extracellular vesicles as potential natural/synthetic non-viral vectors for gene therapy are two of the most exciting developments for the possible application in the field of clinical genetics in the near future. 

The era for high-throughput, single-cell resolution, reliable and highly customizable gene diagnostics platform development is of the utmost importance to advance the success rate of the diagnosis of inheritable diseases, as well to serve as early tools for cancer diagnostics. Once the molecular alterations standing behind a certain phenotype are identified, the reversion and/or prevention of irreversible organ damage are the main targets in these conditions. Currently, one of the biggest challenge of gene therapy is the delivery system. Efficiency, delivery rate, targeting, and safety are the main concerns, for which extracellular vesicles are particularly of high interest. The main relevant topics for this Special Issue are: 

  • Long-read sequencing-based gene diagnostics assays
  • Flow cytometry-based screening/ diagnostics of inherited diseases
  • Extracellular vesicles engineering and molecular characterization for potential gene therapy applications
  • Extracellular vesicles as shuttle vehicles for gene delivery
  • Targeting of extracellular vesicles for gene delivery enhancement

Dr. Árpád Ferenc Kovács
Prof. Dr. Gyorgy Fekete
Guest Editors

Manuscript Submission Information

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Keywords

  • long-read sequencing
  • nanopore-sequencing
  • single-cells
  • extracellular vesicles
  • gene therapy
  • CRISPR-Cas9

Published Papers (7 papers)

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Research

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14 pages, 3898 KiB  
Article
scGENA: A Single-Cell Gene Coexpression Network Analysis Framework for Clustering Cell Types and Revealing Biological Mechanisms
by Yousif A. Algabri, Lingyu Li and Zhi-Ping Liu
Bioengineering 2022, 9(8), 353; https://doi.org/10.3390/bioengineering9080353 - 30 Jul 2022
Cited by 4 | Viewed by 3809
Abstract
Single-cell RNA-sequencing (scRNA-seq) is a recent high-throughput technique that can measure gene expression, reveal cell heterogeneity, rare and complex cell populations, and discover cell types and their relationships. The analysis of scRNA-seq data is challenging because of transcripts sparsity, replication noise, and outlier [...] Read more.
Single-cell RNA-sequencing (scRNA-seq) is a recent high-throughput technique that can measure gene expression, reveal cell heterogeneity, rare and complex cell populations, and discover cell types and their relationships. The analysis of scRNA-seq data is challenging because of transcripts sparsity, replication noise, and outlier cell populations. A gene coexpression network (GCN) analysis effectively deciphers phenotypic differences in specific states by describing gene–gene pairwise relationships. The underlying gene modules with different coexpression patterns partially bridge the gap between genotype and phenotype. This study presents a new framework called scGENA (single-cell gene coexpression network analysis) for GCN analysis based on scRNA-seq data. Although there are several methods for scRNA-seq data analysis, we aim to build an integrative pipeline for several purposes that cover primary data preprocessing, including data exploration, quality control, normalization, imputation, and dimensionality reduction of clustering as downstream of GCN analysis. To demonstrate this integrated workflow, an scRNA-seq dataset of the human diabetic pancreas with 1600 cells and 39,851 genes was implemented to perform all these processes in practice. As a result, scGENA is demonstrated to uncover interesting gene modules behind complex diseases, which reveal biological mechanisms. scGENA provides a state-of-the-art method for gene coexpression analysis for scRNA-seq data. Full article
(This article belongs to the Special Issue Novel Expansions and Trends in Gene Diagnostics and Gene Therapy)
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16 pages, 11100 KiB  
Article
Enhancing Prednisone-Based Arthritis Therapy with Targeted IL-27 Gene Delivery
by Adriana A. Marin, Richard E. Decker, Shreya Kumar, Zachary Lamantia, Hiroki Yokota, Todd Emrick and Marxa L. Figueiredo
Bioengineering 2022, 9(6), 248; https://doi.org/10.3390/bioengineering9060248 - 09 Jun 2022
Cited by 1 | Viewed by 2818
Abstract
Rheumatoid arthritis (RA) is a chronic autoimmune disease which is characterized primarily by synovial hyperplasia and accumulation of several types of immune infiltrates that promote progressive destruction of the articular structure. Glucocorticoids are often prescribed to treat RA because of their strong anti-inflammatory [...] Read more.
Rheumatoid arthritis (RA) is a chronic autoimmune disease which is characterized primarily by synovial hyperplasia and accumulation of several types of immune infiltrates that promote progressive destruction of the articular structure. Glucocorticoids are often prescribed to treat RA because of their strong anti-inflammatory and immunosuppressive effects. However, their application must be limited to the short-term due to a risk of adverse events. In the present study, we examined the potential combination of low-dose prednisone with gene delivery of an agent of promising and complementary effectiveness in RA, interleukin (IL)-27. IL-27 has been shown to have anti-inflammatory potential, while also acting as an effective bone-normalization agent in prior reports. The present report examined a version of IL-27 targeted at the C-terminus with a short ‘peptide L’ (pepL, LSLITRL) that binds the interleukin 6 receptor α (IL-6Rα) upregulated during inflammation. By focusing on this targeted form, IL-27pepL or 27pL, we examined whether the anti-inflammatory potential of prednisone (at a relatively low dose and short duration) could be further enhanced in the presence of 27pL as a therapy adjuvant. Our results indicate that 27pL represents a novel tool for use as an adjuvant with current therapeutics, such as prednisone, against inflammatory conditions. Full article
(This article belongs to the Special Issue Novel Expansions and Trends in Gene Diagnostics and Gene Therapy)
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17 pages, 3202 KiB  
Article
Magnetic Nanoparticles as a Component of Peptide-Based DNA Delivery System for Suicide Gene Therapy of Uterine Leiomyoma
by Sofia Shtykalova, Anna Egorova, Marianna Maretina, Vladislav Baranov and Anton Kiselev
Bioengineering 2022, 9(3), 112; https://doi.org/10.3390/bioengineering9030112 - 08 Mar 2022
Cited by 4 | Viewed by 2120
Abstract
Suicidegene therapy is considered a promising approach for the treatment of uterine leiomyoma (UL), a benign tumor in women characterized by precise localization. In this study, we investigate the efficiency of αvβ3 integrin-targeted arginine-rich peptide carrier R6p-cRGD electrostatically bound to magnetic nanoparticles (MNPs) [...] Read more.
Suicidegene therapy is considered a promising approach for the treatment of uterine leiomyoma (UL), a benign tumor in women characterized by precise localization. In this study, we investigate the efficiency of αvβ3 integrin-targeted arginine-rich peptide carrier R6p-cRGD electrostatically bound to magnetic nanoparticles (MNPs) for targeted DNA delivery into the UL cells. The physico–chemical and cytotoxic properties, transfection efficiency, and specificity of R6p-cRGD/DNA/MNPs polyplexes were evaluated. The addition of MNPs resulted in a decrease in the time needed for successful transfection with simultaneous increase in efficiency. We revealed a therapeutic effect on primary UL cells after delivery of plasmid encoding the herpes simplex virus type 1 (HSV-1) thymidine kinase gene. Treatment with ganciclovir resulted in 20% efficiency of suicide gene therapy in UL cells transfected with the pPTK-1 plasmid. Based on these results, we conclude that the use of cationic peptide carriers with MNPs can be promising for the development of modular non-viral carriers for suicide gene delivery to UL cells. Full article
(This article belongs to the Special Issue Novel Expansions and Trends in Gene Diagnostics and Gene Therapy)
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12 pages, 2728 KiB  
Article
Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9
by Sailan Shui, Shaojie Wang and Jia Liu
Bioengineering 2022, 9(2), 83; https://doi.org/10.3390/bioengineering9020083 - 21 Feb 2022
Cited by 7 | Viewed by 3749
Abstract
The emergence of CRISPR-Cas9 technology has revolutionized both basic and translational biomedical research. For Cas9 nuclease to exert genome editing activity, nuclear localization signal (NLS) derived from simian virus 40 (SV40) T antigen is commonly installed as genetic fusion to direct the intracellular [...] Read more.
The emergence of CRISPR-Cas9 technology has revolutionized both basic and translational biomedical research. For Cas9 nuclease to exert genome editing activity, nuclear localization signal (NLS) derived from simian virus 40 (SV40) T antigen is commonly installed as genetic fusion to direct the intracellular Cas9 proteins to the nucleus of cells. Notably, previous studies have shown that multiple SV40 NLS fusion can improve the targeting activity of Cas9-derived genome-editing and base-editing tools. In addition, the multi-NLS fusion can increase the intracellular activity of Cas9 in the forms of both constitutive expression and directly delivered Cas9-guide RNA ribonucleoprotein (RNP) complex. However, the relationship between NLS fusion and intracellular Cas9 activity has not been fully understood, including the dependency of activity on the number or organization of NLS fusion. In the present study, we constructed and purified a set of Streptococcus pyogenes Cas9 (SpCas9) variants containing one to four NLS repeats at the N- or C-terminus of the proteins and systematically analyzed the effects of multi-NLS fusion on the activity of SpCas9 RNPs. It was found that multi-NLS fusion could improve the intracellular activity as lipofected or nucleofected Cas9 RNPs. Importantly, multi-NLS fusion could enhance the genome-editing activity of SpCas9 RNPs in primary and stem/progenitor cells and mouse embryos. Full article
(This article belongs to the Special Issue Novel Expansions and Trends in Gene Diagnostics and Gene Therapy)
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15 pages, 4910 KiB  
Article
A Second-Generation Nanoluc-IL27 Fusion Cytokine for Targeted-Gene-Therapy Applications
by Janelle Wesleyn Salameh, Shreya Kumar, Cosette Marie Rivera-Cruz and Marxa Leao Figueiredo
Bioengineering 2022, 9(2), 77; https://doi.org/10.3390/bioengineering9020077 - 16 Feb 2022
Cited by 5 | Viewed by 2520
Abstract
An emerging approach in treating skeletal malignancies utilizes osteoimmunology to investigate new multifunctional immune-stimulatory agents that can simultaneously combat tumor growth and promote bone repair. We have hypothesized that cytokine Interleukin-27 (IL-27) is an excellent candidate biologic to help rebalance the prostate tumor [...] Read more.
An emerging approach in treating skeletal malignancies utilizes osteoimmunology to investigate new multifunctional immune-stimulatory agents that can simultaneously combat tumor growth and promote bone repair. We have hypothesized that cytokine Interleukin-27 (IL-27) is an excellent candidate biologic to help rebalance the prostate tumor cells and bone cell environment. In this work, we examined the proof of principle for a short, secreted luciferase (Nanoluc or Nluc) fusion with IL-27 to produce a novel cytokine-based biologic (Nluc-27), whereby we examined its efficacy in vitro in reducing prostate tumor growth and rebalancing bone cell proliferation and differentiation. This work demonstrates the targeting and anti-tumor efficacy of the Nluc-27 fusion cytokine in cancer and bone cell models. The fusion cytokine is detectable in conditioned media, and bioactive in different cell systems. This novel Nluc-27 cytokine will allow flexible incorporation of other targeting domains and may serve as flexible tool to augment IL-27′s bioactivity and reengineer its efficacy against prostate tumor or bone cells, and may prove applicable to several other cell types for targeted gene therapy applications. Full article
(This article belongs to the Special Issue Novel Expansions and Trends in Gene Diagnostics and Gene Therapy)
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25 pages, 4836 KiB  
Article
Octreotide-Targeted Lcn2 siRNA PEGylated Liposomes as a Treatment for Metastatic Breast Cancer
by Vrinda Gote and Dhananjay Pal
Bioengineering 2021, 8(4), 44; https://doi.org/10.3390/bioengineering8040044 - 03 Apr 2021
Cited by 10 | Viewed by 4649
Abstract
Lcn2 overexpression in metastatic breast cancer (MBC) can lead to cancer progression by inducing the epithelial-to-mesenchymal transition and enhancing tumor angiogenesis. In this study, we engineered a PEGylated liposomal system encapsulating lipocalin 2 (Lcn2) small interfering RNA (Lcn2 siRNA) for selective targeting MBC [...] Read more.
Lcn2 overexpression in metastatic breast cancer (MBC) can lead to cancer progression by inducing the epithelial-to-mesenchymal transition and enhancing tumor angiogenesis. In this study, we engineered a PEGylated liposomal system encapsulating lipocalin 2 (Lcn2) small interfering RNA (Lcn2 siRNA) for selective targeting MBC cell line MCF-7 and triple-negative breast cancer cell line MDA-MB-231. The PEGylated liposomes were decorated with octreotide (OCT) peptide. OCT is an octapeptide analog of somatostatin growth hormone, having affinity for somatostatin receptors, overexpressed on breast cancer cells. Optimized OCT-targeted Lcn2 siRNA encapsulated PEGylated liposomes (OCT-Lcn2-Lipo) had a mean size of 152.00 nm, PDI, 0.13, zeta potential 4.10 mV and entrapment and loading efficiencies of 69.5% and 7.8%, respectively. In vitro uptake and intracellular distribution of OCT-Lcn2-Lipo in MCF-7 and MDA-MB-231 and MCF-12A cells demonstrated higher uptake for the OCT-targeted liposomes at 6 h by flow cytometry and confocal microscopy. OCT-Lcn2-lipo could achieve approximately 55−60% silencing of Lcn2 mRNA in MCF-7 and MDA-MB-231 cells. OCT-Lcn2-Lipo also demonstrated in vitro anti-angiogenic effects in MCF-7 and MDA-MB-231 cells by reducing VEGF-A and reducing the endothelial cells (HUVEC) migration levels. This approach may be useful in inhibiting angiogenesis in MBC. Full article
(This article belongs to the Special Issue Novel Expansions and Trends in Gene Diagnostics and Gene Therapy)
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Review

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13 pages, 1759 KiB  
Review
Enhancing Molecular Testing for Effective Delivery of Actionable Gene Diagnostics
by Árpád Ferenc Kovács, Zaránd Némethi, Tünde Abonyi, György Fekete and Gábor T. Kovács
Bioengineering 2022, 9(12), 745; https://doi.org/10.3390/bioengineering9120745 - 01 Dec 2022
Viewed by 1390
Abstract
There is a deep need to navigate within our genomic data to find, understand and pave the way for disease-specific treatments, as the clinical diagnostic journey provides only limited guidance. The human genome is enclosed in every nucleated cell, and yet at the [...] Read more.
There is a deep need to navigate within our genomic data to find, understand and pave the way for disease-specific treatments, as the clinical diagnostic journey provides only limited guidance. The human genome is enclosed in every nucleated cell, and yet at the single-cell resolution many unanswered questions remain, as most of the sequencing techniques use a bulk approach. Therefore, heterogeneity, mosaicism and many complex structural variants remain partially uncovered. As a conceptual approach, nanopore-based sequencing holds the promise of being a single-molecule-based, long-read and high-resolution technique, with the ability of uncovering the nucleic acid sequence and methylation almost in real time. A key limiting factor of current clinical genetics is the deciphering of key disease-causing genomic sequences. As the technological revolution is expanding regarding genetic data, the interpretation of genotype–phenotype correlations should be made with fine caution, as more and more evidence points toward the presence of more than one pathogenic variant acting together as a result of intergenic interplay in the background of a certain phenotype observed in a patient. This is in conjunction with the observation that many inheritable disorders manifest in a phenotypic spectrum, even in an intra-familial way. In the present review, we summarized the relevant data on nanopore sequencing regarding clinical genomics as well as highlighted the importance and content of pre-test and post-test genetic counselling, yielding a complex approach to phenotype-driven molecular diagnosis. This should significantly lower the time-to-right diagnosis as well lower the time required to complete a currently incomplete genotype–phenotype axis, which will boost the chance of establishing a new actionable diagnosis followed by therapeutical approach. Full article
(This article belongs to the Special Issue Novel Expansions and Trends in Gene Diagnostics and Gene Therapy)
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