Design of non-viral vector with improved regulatory features towards therapeutic application

Viral vectors based gene therapy is often compromised by adverse immunological reactions raising safety concerns. Hence, improved design and development of non-viral vectors with strong regulatory regions is desired. We describe the design of a non-viral mammalian expression vector in which the primary transgene (a truncated dystrophin gene linked with Duchenne muscular dystrophy (DMD)) named microdystrophin delR4-R23/delCT (MD1) is under the transcriptional control of elements of desmin locus control region (DES-LCR). The designed vector, named as DES-LCR/MD1-EGFP, was constructed by cloning two fragments into the pBluescript backbone. Fragment 1 contains DES-LCR enhancer and DES-LCR promoter region while fragment 2 contains MD1 transgene and reporter EGFP (enhanced green fluorescent protein) gene separated by linker P2A (2A peptide). This vector design provides a framework for strong regulation with non-viral features. This design forms the foundation for application in conditions linked to multisystem diseases.


Background:
Applications of gene therapy are tremendous and offers hope to treat genetic diseases at large. Success of gene therapy lies on the design, development and delivery of vectors, which can be of viral and non-viral types. Both, viral and non-viral gene therapy vectors, have applications in preclinical and clinical settings. Viral vectors have emerged as effective gene therapy vehicles for clinical gene therapy [1], however, safety has been an issue on use of viral vectors since they may generate strong immune response [2]. On the other hand, non-viral vectors have been ignored in the past but they certainly represent the long-term future of gene therapy owing to their increased safety. In recent years, interest towards ©Biomedical Informatics (2020) development of non-viral DNA vectors has progressed steadily [3], and several non-viral vector systems have been developed and successfully employed for safe delivery [4]. There are opportunities for further development of non-viral vectors with improved design to address therapeutic needs. One of the solutions to improve the design includes inclusion of strong regulatory features to achieve desired expression of transgenes.
Role of cis-regulatory elements, as strong regulatory sequences, in the design of gene therapy vectors has long been known [5][6]. Sequences of locus control regions (LCRs), non-coding cisregulatory regions, have been used in the past in viral vectors to control expression of transgenes [7][8]. Ability to control gene expression at ectopic sites makes LCRs unique and different from other distal elements of the genome. Unique regulatory signatures have been identified in the human LCR sequences [9], and further efforts to use these special regulatory elements in the design of gene therapy vectors will pave the way to generate new generation of gene therapy vectors with increased safety.
It is of interest to design a non-viral mammalian expression vector for the expression of a truncated version of dystrophin gene, microdystrophin delR4-R23/delCT (MD1). Typically, truncated versions of dystrophin gene are preferred choice for gene therapy of DMD [10]. Despite the promise of gene therapy for the treatment of DMD, it has been challenging to achieve optimum and prevalent expression of therapeutic gene [11]. Hence, we describe the design of a non-viral vector with improved regulatory features using computer aided sequence analysis tools Methodology: Vector design: SnapGene v5.0 software (from GSL Biotech; available at snapgene.com) was used to rationally design a non-viral mammalian expression vector containing elements of human LCR (DES-LCR) for expression of transgenes (MD1 and EGFP). pBluescript plasmid (3931bp) (adapted from Addgene plasmid #24595) was used as empty backbone to construct the vector. Features of this empty backbone are listed in Table 1.

Amplification of MD1 gene:
From the constructed DES-LCR/MD1-EGFP vector sequence, forward and reverse primers, MD1 F (24bp) and MD1 R (29bp) respectively, were designed using the 'add primer' option of SnapGene for the in-silico amplification of MD1 gene. Restriction sites were added in the primers for future cloning of the MD1 gene. MD1 F contain EcoRV restriction site and MD1 R contain HindIII restriction site (Table 2). 'PCR' feature of SnapGene was used to amplify the MD1 gene from the constructed DES-LCR/MD1-EGFP vector. MD1 F and MD1 R primers were selected and in-silico PCR was run. Further, 'simulate agarose gel' option was used to confirm the size of amplified MD1 gene on 1% agarose.

Translation of transgenes:
MD1 and EGFP transgenes were translated to generate amino acid products using the 'translation feature' of SnapGene and HindIII are also present at the start and end, respectively, of the amplified sequence for future cloning purposes.

Results: Designed DES-LCR/MD1-EGFP vector:
Non-viral mammalian expression vector, constructed by cloning two different fragments into the pBluescript backbone using SnapGene, was named as 'DES-LCR/MD1-EGFP' vector. DES-LCR/MD1-EGFP is an 8588bp long circular vector characteristics of which are given in Table 3. DES-LCR/MD1-EGFP is a high copy number vector for growth in bacteria and contains Ampicillin resistance gene, which confers resistance to antibiotic Ampicillin for selection during bacterial growth. Polyadenylation signal from SV40 poly(A) sequence of the vector helps in addition of poly(A) tail to generated mRNAs from the transgenes during propagation in mammalian cells.

Discussion:
With viral vectors dominating cell and gene therapy, non-viral vectors sidestep the main concerns that come with using viruses: safety, immunogenicity and manufacturing limits (yield, scaling-up and costs) [13]. New generation of vectors with increased safety are desirable for gene therapy of constitutional disorders to achieve permanent genetic modification and stable expression of transgenes. Introduction of novel and strong regulatory elements in the non-viral vectors provides a solution towards improved design of gene therapy vectors.
LCRs are unique non-coding regulatory sequences with their ability to control gene expression at ectopic locations. These regulatory sequences have not been studied much in the past, however, their presence in the mammalian genomes makes them important. LCRs have the ability to enhance the expression of linked genes to physiological levels indicating that they play a significant role in controlling the expression of target genes [14]. Potential  DES-LCR/MD1-EGFP is a non-viral mammalian expression vector and hence a safer option over use of viral vectors expressing dystrophin gene for applications in muscle-directed gene therapy. Functional studies will be needed to validate the performance and usefulness of this vector in therapeutic settings. In recent past, novel designs of non-viral vector systems have been described with promising applications [24]. The designed vector in this work is another step towards tackling, potentially, the current challenges of vector design, which may prove to be useful in future, studies for treating multisystem diseases. Use of non-viral vectors comprising of human elements have been suggested ideal for human gene therapy as they deliver sustainable therapeutic levels of gene expression without adverse immunological effects [25]. Human LCRs are strong regulatory elements, hence, can be considered as preferred choice to construct additional regulatory systems for their applications in biomedical research. Ultimately, these vectors will inflate the traditional applications of gene therapy and will also lead towards newer other opportunities in the field of basic science and clinical research.

Conclusion:
We describe the design of non-viral vector with improved regulatory features using computer aided sequence analysis tools. This forms a framework towards design of new generation of gene therapy vectors with increased safety. It should be noted that this design should be validated with adequate experimental data.

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