Strategies for conjugating fluorescent dyes to plasmid DNA to visually track vectors used in gene therapy for cancer or other diseases

 Various gene therapies have been approved by FDA (U. S. Food & Drug Administration).  To treat cancer (ex. multiple myeloma, diffuse large B-cell lymphoma), several immunotherapies (ex. Novartis, Johnson & Johnson) have been approved since 2017, which involve administering T lymphocytes obtained from patients that have been genetically engineered to express 'chimeric antigen receptor' (CAR) recognizing tumor-specific proteins (https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products).  In addition, virus therapy (Amgen), which involves injecting (into tumors) modified herpesvirus designed to replicate and rupture cancer cells selectively, has been approved to treat melanoma in 2015 (Fukuhara et al., 2016).  For treating 'Leber's congenital amaurosis' (inherited retinal condition progressively leading to blindness), recombinant AAV (adeno-associated virus), which has been modified to express the cDNA encoding RPE65 (retinal pigment that mediates phototransduction), has been approved in 2017 (Clarke et al., 2017).  To treat the neuromuscular disease 'spinal muscular atrophy', infusion (intravenous) of AAV9 virus (Novartis; 2.1M USD) that has been engineered to express SMN1 gene (functions in the biogenesis of splicing complex) was approved in 2019 (Mendell et al., 2021).

 However, the clinical use of viral vectors has faced several limitations.  Despite their ability to infect and transfer the genetic payload, the potential for insertional mutagenesis (ex. retrovirus, lentivirus) exists.  Earlier gene therapy trials of SCID (severe combined immunodeficiency caused by adenosine deaminase deficiency) patients documented the integration of the retroviral vector at an oncogenic locus in hematopoietic progenitor cell genome (Fischer et al., 2019).  Further, triggering immunological response may exclude the use of viruses (ex. adeno-associated virus type 2, adenovirus serotype 5) or limit repetitively administring viral vector (ex. adenovirus).  This has inspired pharmaceutical interest in using plasmids as a potential non-viral vector for gene therapy.

 The use of plasmids as gene delivery vector offers several advantages: easy to design, longer stability, inexpensive, less damaging to cells, minimal rate of genome integration, etc.  Nevertheless, the disadvantages include inefficient gene delivery, potential breakage of plamids, depletion of plasmids due to cell division, and immune recogntion of unmethylated bacterial CpG sequences.  It entailed making extensive modifications to plasmids to facilitate clinical application (Hardee et al., 2017).  For instance, to meet the FDA approval, the antibiotics-resistance gene used for selection had to be removed (to preempt dissemination into the environment).  As a substitute, plasmids harboring DNA sequences that titrate out repressors, which inhibit the replication of host bacterium by binding to the operator of ori (origin of replication), were developed (Marie et al., 2010).  Alternatively, plasmids expressing suppressor tRNA for the host bacteria (conditional mutant for ori) were designed. (Lilly et al., 2015).  Several plasmid-based gene therapies for infectious diseases (ex. HIV, HPV, hepatitis, malaria, influenza) or other disorder (ex. cardiac disease, cancer, diabetes) have progressed to phase I or II clinical trials (Hardee et al, 2017).

 Inefficient delivery could result in reduced drug activity as well as undesirable side effects.  Hence, equally significant for the development of plasmid-based gene delivery vectors is the ability to track them.  To trace, plasmids could be directly labeled with dyes to visually determine their location.  Ideally, labeling should occur without causing the degradation or altering the conformation of plasmids. 

For random direct labeling (not sequence directed), a previously developed method involves covalently labeling plasmid DNA via alkylating at the N⁷ position of (deoxy)guanine.  In Mirus 'Label IT' kit, a short linker was used to label DNA with a fluorophore.  Labeling of plasmids with the fluorscent dye cyanine using this approach did not affect the expression of the encoded genes (Watt et al. 2002) though it may affect transfection efficiency, endosomal escape, dissociation of DNA from a carrier, or nuclear retention of plasmid DNA.  In ULYSIS Nucleic Acid Labeling Kit (Molecular Probe), one of the coordination sites of the platinum-fluorophore complex was used for conjugating to the N⁷ position of (deoxy)guanine.   

An alternative method by Delvaux et al. (University of Iowa, USA) involves labeling plasmid DNA via forming a complex with 'cationic peptide-fluorphore' (containing aryl azide photolabel), followed by photoactivation (Devalux et al., 2022).  In one of their constructs, photolabel was added at the N-terminus while the sulfo-Cy5 fluorophore was conjugated to the C--teminus of NLS (nuclear localization signal) peptide from SV40 (simian virus 40)'s large T antigen for initial electrostatic interaction with DNA (double stranded).  In Photoprobe or FastTag Nucleic Acid Labeling System (Vector Laboratories), photoaffinity labeling was used to introduce fluorophore, biotin, primary amine, or a disulfide bond [upon reduction, it could react with thiol-reactive reagents, ex. maleimide-coupled fluorophore, hapten (detected using antibody)] (Watt et al. 2002). 

Other methods include incorporation of fluorophore or hapten-labeled nucleotide by DNA polymerase I (ex.  'nick translation'), TdT (terminal deoxynucleotidyl transferase), DNA ligase, DNA polymerase I's Knenow fragment (ex. fill-in reaction), etc.   For non-covalent labeling, DNA intercalating dyes such as DAPI or Hoechst 33258 could be used.   For efficiency, click-chemistry could be utilized by reacting alkyne [or dibenzocyclooctyne (DBCO)]-containing nucleotide (after incorporation) with azide-modified fluorophore, for instance [Rombouts et al., 2016).

For sequence-specific labeling, enzymes could be used to directly modify DNA.   DNA methyltransferases (MTases) could be used to catalyze the addition of fluorophore or functional groups (ex. alkenyl, alkynyl) (Rombouts et al., 2016; Deen et al., 2018).  Alternatively, fluorophore-conjugated oligonucleotide could hybridize to a specific complementary sequence [ex. triple-forming oligonucleotide (Molecular Beacons) utilizing FRET (fluorescence resonance energy transport)].  Or, proteins binding to specific DNA (or RNA) sequence (ex. transcription factors like LacI) fused to GFP (green fluorescence protein) could be expressed intracellularly to track administered polynucleotide. 

For vaccination, both plasmid DNA and mRNA have been developed.  For mRNA vaccine,  recognition by the innate system (TLR7, TLR8, TLR3) or cytoplasmic signaling [ex. RIG-1, Melanoma Differentiation-Associated protein 5 (MDA5), 2'-5'-oligoadenylate synthetase 1 (OAS), interferon-induced, double-stranded RNA-activated protein kinase (PKR)] entailed substituting with modified nucleosides.  Purification of other components associated with generating mRNA (ex. DNA template, unused nucleoside, double stranded RNA) is necessary to obviate immunogenicity, which could dampen antigen translation efficacy.  For repeated administration, potential issues associated with utilizing unnatural modified nucleosides or delivery vectors may need to be considered.  Compared to mRNA vaccine, plasmid DNA-based vaccine is lesser immunogenic and the concern over genomic integration or inciting autoimmune response against DNA has largely been subsided (Liu, 2019).  Other advantages of using plasmid DNA include greater stability, amplification via transcribing multiple mRNAs from each plasmid, simpler and speedier production, less costly, etc. albeit plasmid DNA must travel to the nucleus to generate mRNA.  A 'minimalistic, immunologically defined gene expression' (MIDGE) vector was used to express hTNF (human tumor necrosis factor) to sensitize melanoma cells to the anticancer drug vindesine (Kobelt et al., 2014).

The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation.  For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as to conjugate to peptidesor antibodies. It provides custom conjugation of small molecules such as chemical drugs, metabolites and labeled compounds with synthetic or natural polymers (enzymes, peptide, protein, oligonucleotide, antibody, dendrimer, nanoparticle, etc).  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  It has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

https://www.biosyn.com/oligo-flourescent-labeling.aspx

https://www.biosyn.com/tew/Speed-up-Identification-of-COVID19.aspx

https://www.biosyn.com/covid-19.aspx

https://www.biosyn.com/mrna.aspx

https://www.biosyn.com/bioconjugation.aspx

https://www.biosyn.com/tew/Drug-Conjugation-Synthesis-Strategies.aspx

https://www.biosyn.com/tew/Basic-Bioconjugation-Chemistry-of-Reactive-Groups-in-Biomolecules.aspx

https://www.biosyn.com/tew/Maleimide-labeling-of-thiolated-biomolecules.aspx

References

Clarke T. Gene Therapy for Blindness Appears Initially Effective, Says U.S. FDA. Scientific American. October (2017).

Deen J, Neely RK. et al. A general strategy for direct, enzyme-catalyzed conjugation of functional compounds to DNA.  Nucleic Acids Res.  46 :e64 (2018).  PMID: 29546351

Delvaux NA, Mathew B, Rice KG. Fluorescent labeling of plasmid DNA for gene delivery: Implications of dye hydrophobicity on labeling efficiencies and nanoparticle size.  Anal Biochem. 2022 May 1;644:113895.  PMID: 32783899

Fischer A, Hacein-Bey-Abina S. Gene therapy for severe combined immunodeficiencies and beyond. J Exp Med.  217:e20190607 (2020).  PMID: 31826240

Rombouts K, Remaut K.  Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies.   Bioconjug Chem.  27:280-97 (2016).  PMID: 26670733

Fukuhara H, Ino Y, et al.  Oncolytic virus therapy: A new era of cancer treatment at dawn.  Cancer Sci. 107:1373-1379 (2016).  PMID: 27486853

Hardee CL, Arévalo-Soliz LM, et al. Advances in Non-Viral DNA Vectors for Gene Therapy.  Genes (Basel) 8 :65 (2017).  PMID: 28208635

Kobelt D, Walther W. et al. Preclinical study on combined chemo- and nonviral gene therapy for sensitization of melanoma using a human TNF-alpha expressing MIDGE DNA vector.  Mol Oncol. 8: 609-19 (2014).  PMID: 24503218

Lilly, J.; Camps, M. Mechanisms of theta plasmid replication. Microbiol. Spectr. 3, 1–18 (2015). PMID: 26104556

Liu MA.  Comparison of Plasmid DNA and mRNA as Vaccine Technologies.  Vaccines (Basel). 7:37 (2019).  PMID: 31022829

Marie, C.; Scherman, D. pFARs, plasmids free of antibiotic resistance markers, display high-level transgene expression in muscle, skin and tumour cells. J. Gene Med. 12, 323–332 (2010).  PMID: 20209487

Mendell JR, Al-Zaidy SA, et al. Current Clinical Applications of In Vivo Gene Therapy with AAVs.  Mol Ther.  29:464-488 (2021).  PMID: 33309881

Watt M, Hagstrom JE.  Intracellular Localization and Expression of Labeled Plasmid DNA using  Label IT Tracker™ Nucleic Acid Labeling Kits.  Technical Report(Mirus). (2002).