Cell-penetrating Peptides (CPPs) for Gene Therapy
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What is the gene therapy?
One novel way to cure or prevent disease is through gene therapy, which involves changing a person's genes in their cells. Specifically, it seeks to treat hereditary and mutation-based genetic abnormalities that cause illness. In order to cure a specific condition, gene therapy involves transferring genetic material (DNA or RNA) to repair, control, or replace genes. From its conceptual beginnings in the treatment of single-gene disorders like cystic fibrosis and hemophilia, gene therapy has expanded to encompass a broader range of approaches, such as methodologies for inducing cell death or producing therapeutic proteins in vivo, as well as their clinical investigations in a variety of inherited and acquired diseases. Cancers include breast, uterine, and lung can benefit greatly from gene therapy when combined with other treatment techniques including radiation, chemotherapy, or immune activation. Improving the efficiency of gene transport into the target tissue is a major hurdle in the gene therapy technique. Therefore, a molecular carrier or gene delivery vehicle, sometimes referred to as a "vector," is utilized to ensure the effective expression of the target gene in the cell. The use of vectors, both viral and non-viral, has led to the development of several gene transfer delivery methods that can transport the transgene into specific cells.
Schematic of different gene therapy approach. (Henderson M L., et al., 2024)
Significance of cell-penetrating peptides in gene therapy
When it comes to gene therapy, cell-penetrating peptides (CPPs) are crucial because they improve the uptake of genetic materials into cells. Conjugation with plasmid DNA, siRNA, mRNA, and antisense oligonucleotides are just a few examples of the nucleic acid forms that may be attached to these, allowing for the efficient delivery of therapeutic genetic resources to specific cells. Encapsulating or binding to nucleic acids allows CPPs to enhance their stability and bioavailability by protecting them from destruction by nucleases in biological settings. When it comes to gene therapy, the extracellular matrix and cell membranes are two of the physiological hurdles that may be overcome with the aid of CPPs. To get insights into the therapeutic effectiveness, CPPs can be engineered to incorporate fluorescent tags or other markers that enable real-time tracking of gene transport and expression within living organisms.
Strategy of CPPs conjugated to nucleic acids
There are two major ways that cell-penetrating peptides (CPPs) are usually created to be conjugated to nucleic acids: covalently and noncovalently. A variety of stable linkers, including oxime, amide, and thiazolidine bonds, as well as cleavable chemical linkers, such as disulfide and hydrazine linkage, allow CPPs to covalently conjugate with nucleic acids. Under appropriate environmental circumstances, cleavable bonds like disulfide and hydrazine may be readily broken, releasing the oligonucleotide from the CPP and allowing genes to be delivered to specified cells or tissues. To improve the transport efficiency, this approach always incorporates PEG polymer as a crosslinker. PEG polymer can decrease electrostatic interactions between positively charged peptides and negatively charged nucleic acids. To accomplish their biological activities, CPPs and nucleic acids can be stablely conjugated using chemical covalent linkers such as oxime, amide, or thiazolidine bonds. This allows the conjugates to pass through cell membranes and reach the nucleus. Technical challenges in chemical conjugation and purification of conjugates, as well as concerns about potential changes to the biological activity of nucleic acids, limit the use of covalent conjugation to uncharged oligonucleotide analogs such as peptide nucleic acid (PNA) and phosphonodiamidate morpholino oligomer (PMO).
Consequently, a new method for delivering nucleic acids based on CPP has been developed and put into use: noncovalent conjugation. This method relies on electrostatic interactions and is thus more suited for the transport of large negatively charged nucleic acids such as plasmid DNA, siRNA, and mRNA. Nucleic acid incorporation into CPP-nanoparticles (NP) conjugates and direct contact with nucleic acids by coincubation under suitable circumstances are the two known ways of CPP-mediated non-covalent conjugation. Short peptides that can mix with nucleic acids without further chemical changes or cross-linking constitute the basis of the first strategy, which is straightforward to make. The second method involves the incorporation of nucleic acids into nanoparticles (NP) by electrostatic interactions; it is used to create functionalized peptides with nanoparticles such as dendrimers, micelles, lipids, or quantum dots.
A variety of CPP-based nucleic acid delivery techniques and their applications. (Geng J., et al., 2022)
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CPP based DNA delivery
The vast majority of an organism's genetic material is dsDNA, which includes the blueprints for every protein the body makes. Achieving high transfection efficiency is crucial for using DNA in pharmaceutical or fundamental research applications. This is aided by efficient DNA delivery, which involves DNA internalization, and efficient DNA expression, which involves target DNA transcription and subsequent translation. There are some unsavory safety issues associated with viral vectors, the most used DNA delivery technology at the moment, despite their tremendous effectiveness. Potential future options for DNA delivery that aim to circumvent these restrictions include non-viral technologies with improved safety profiles. Research on CPP, a non-viral delivery technology that uses strong electrostatic interactions to conjugate nucleic acids, has led to its application in DNA delivery. In vitro delivery of EGFP-encoding plasmids, using the amphipathic peptide MGPE9 (CRRLRHLRHHYRRRWHRFRC), following delivering reporter plasmids, the nanocomplexes showed widespread and long-lasting gene expression in skin tissue following in vivo administration, with no discernible side effects or harmful consequences. The α-helical structure of CVP1, which is derived from chicken anemia virus (CAV) and shares similarities with HIV-TAT, was shown to be able to transport the plasmid pCDNA3.1-RFP into HCT116 cells. There was a substantial increase in fluorescence compared to the TAT/RFP sample, indicating that CVP1 had a better transfection effectiveness than the TAT peptide. Aside from their use in reporter plasmid delivery, CPPs are also being investigated for potential use in DNA vaccine delivery to combat viral infections. This was proved by the team led by Bolhassani, who created CPP-MPG condensed DNA complexes that encode core components (core E1 and E2 of hepatitis C virus and Human papillomavirus (HPV) E7 oncogene, which is crucial for viral infection). Neither in vitro nor in vivo did the complexes show any signs of cytotoxicity. In contrast to mice injected with control DNA constructions, animals immunized with these complexes exhibited a combination of IgG1 and IgG2a responses, as well as a higher IFN-gamma response. Also, when comparing HPVE7 oncogene vaccination with E7 DNA immunization alone to that with MPG/DNA nanoparticles, tumor development was significantly reduced. A growing body of literature also points to the development of DNA delivery systems based on CPPs as a potential anti-cancer therapy. One study investigated the possibility of using the CPP-RALA peptide to transfer pDNA encoding p53. Gene release and p53 protein expression are facilitated by in-vitro transfection of HeLa cells using RALA/pDNA vectors, which promote apoptosis in cancer cells. Jun et al. developed Tat-E7/pGM-CSF, a peptide-based nanoparticle HPV16 vaccine, which significantly enhanced immune response in vitro and in vivo, suppressed tumor development, and extended long-term life in mice models.
CPP-based DNA delivery. (Geng J., et al., 2022)
CPPs as the novel vectors for gene therapy. (Taylor R E., et al., 2020)
CPP-based mRNA delivery
The messenger RNA (mRNA) is both a byproduct of DNA transcription and a building block for proteins. Because mRNA performs its activities in the cytoplasm rather than the nucleus, making transfection easier, mRNA-based therapies have emerged as a promising option for the future of biomedicine. Since their ability to mix mRNA with molecules of lower charge density form stable nanocomplexes with high membrane-penetrating capabilities, cationic CPPs can be a great vehicle for mRNA delivery. Researchers have shown that CPP Xentry protamine fusion peptides (XP) can transmembrane regulators and reporter genes enter cells. High levels of reporter protein expression were seen in both two-dimensional tissue cultures and three-dimensional cancer cell spheroids in a separate study that demonstrated that a cationic CPP PepFect 14 (PF14) effectively delivered mRNA encoding reporter by generating CPP-mRNA nanoparticles. Impressively, in vivo, PF14 nanoparticles induced expression in many cell types in tumor-associated tissue, surpassing the performance of a lipid-based transfection agent. Udhayakumar's team looked at the possibility of an amphipathic RALA motif-containing CPP delivering antigen-encoding mRNA to the immune system. To facilitate mRNA release from endosomes, RALA compresses mRNA into nanocomplexes that have membrane disrupting characteristics that are pH dependent. RALA/mRNA complexes caused a substantial increase in OVA-specific T cell proliferation and showed effective production of transported mRNA in dendritic cells. It has been found that poly (lactic acid) nanoparticles (PLA-NPs) may stimulate effective immune responses to various model antigens, which gives them an advantage in the field of vaccines. As a result, one study employed cationic CPPs and PLA-NPs as mRNA vector condensing agents. In comparison to naked mRNA, complexes containing CPP-LAH4-L1 and PLA-NPs were able to activate the humoral immune response, which included a prominent Th1 bias, activate innate immunity through pattern recognition receptors, and induce the highest protein expression in dendritic cells (DCs).
CPP-based siRNA delivery
Numerous medicines based on small interfering RNAs are now being tested in human clinical trials to combat various illnesses, such as cancer and viral infections. The use of CPP as a delivery method for siRNA has also shown promising results. It wasn't until 2003 that the idea of using CPP to distribute siRNAs was first proposed. Researchers found that a 60% reduction in target gene expression was achieved when MPG facilitated the transport of siGAPDH and electrostatically condensed siRNA into complexes. Nevertheless, an 80% decrease in GAPDH expression level was achieved by its mutant MPG-ΔNLS, which amplified the RNAi impact. In addition, in vivo applications of MPG-ΔNLS/siRNA complexes have been made to induce gene silencing by delivering siRNA targeting Oct-4 into mouse blastocysts. Additionally, T9(dR) demonstrated minimal toxicity and excellent efficiency in transporting siRNA into many cell lines. All PR8-infected mice in the 50 nmol T9(dR)/siNP group survived tail vein injection and regained their weight two weeks later, according to in vivo studies. A CPP-DRBD fusion protein constituted the foundation of a significant advancement in the CPP-mediated method of siRNA administration. The efficient RNAi response was triggered by packaging siRNA into complexes using CPP-DRBD. A comparable chimeric delivery method for siRNA distribution was recently developed. Nanocomplexes containing siGAPDH can be self-assembled by fusion peptides that include SPACE and cationic oligoarginine. Without obvious tissue damage or immune cell infiltration, the nanocomplexes showed substantial gene silencing effects in vitro and in vivo.
CPP-based siRNA delivery. (Geng J., et al., 2022)
CPPs-based miRNA delivery
The miRNAs control endogenous gene expression via binding to the 3' untranslated regions of mRNAs and either suppressing post-transcriptional gene expression or causing destruction of their targets. These RNAs include 20-30 nucleotides. There is evidence that genetic, cancer, neurological, cardiovascular, infectious, and inflammatory illnesses can all be caused by dysregulation of microRNAs. Restoring their expression and function requires miRNA and mimics, which were created because decreasing miRNA expression might drive the start and progression of illnesses in some cases. However, ensuring the effective and safe transport of miRNA into tissues and cells remains a key barrier for miRNA-based therapeutics. A CPP-based delivery system may be a suitable choice from this point of view. A research found that miR-122 may be safely delivered into C. elegans, mice primary hepatocytes, and mouse liver cells via MPG. No cytotoxicity was observed. In addition, the results of the study demonstrated that the miR-122 mimic provided by MPG and its mutant MPGΔNLS successfully controlled cholesterol levels in mouse hepatocytes. However, the adjustment of miR-122 levels in cells treated with MPGΔNLS/miR-122 complexes was greater than in the MPG/miR-122 group, even when the two groups used the same molar ratio. In addition, PF6 was shown to penetrate human primary keratinocytes and form nanocomplexes with miR-146a mimic, which block the expression of target genes related to the NF-κB pathway. Hence, PF6/miR-146a nanocomplexes reduced ear-swelling and decreased production of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24, and CXCL1 when administered subcutaneously prior to irritating contact dermatitis in a mouse model. To create peptide conjugates using R9, a targeting peptide cRGDfK was added, similar to CPP-delivered siRNA. Through interactions between ligands and receptors, complexes containing the cRGDfK-R9 peptide and miRNA were generated, allowing for the targeted transport of miR-34a into U87MG cells and HeLa cells that express the RGD receptor and the folate receptor, respectively.
CPP-based antisense oligonucleotide (ASO) delivery
ASOs are purpose-built to complement the target sequence; they are single-stranded DNA-like sequences that typically range in length from twelve to twenty-five nucleotides (oligonucleotides). These ASOs can bind to their target sequence with little off-target pairing through Watson-Crick pairing if designed properly. The action mechanism of ASOs is controlled by their chemistry and the specific RNA target binding site.
Muscles all across the body deteriorate in the hereditary disease known as Duchenne muscular dystrophy (DMD). Phosphorodiamidate morpholino oligomer (Exon skipping) is an FDA-approved, potentially life-changing treatment for Duchenne muscular dystrophy (DMD). The research group led by Yokota directly coupled the DG9 peptide to exon-skipping PMO in order to make exon-skipping treatment more effective. DG9 came from the human Hph-1 transcription factor's protein transduction domain and is a CPP. In skeletal muscles, DG9-conjugated PMO demonstrated a skipping efficiency that was 2.2 to 12.3 times greater than unconjugated PMO, and in the heart, the difference was 14.4 times larger, after retro-orbital injection into hDMDdel52; mdx mice. Skeletal muscles showed dystrophin production ranging from 2.8% to 3.9% and an exon 51 skipping level ranging from 55 to 71% after three weeks of weekly DG9-PMO injections. Following injection, DG9-PMO did not cause any noticeable side effects. The efficacy and restoration of dystrophin following intramuscular injection of DG9-PMO into the tibialis anterior muscle further supports the drug's promise as a treatment for Duchenne muscular dystrophy.
In a similar vein, Bersani et al. used CPP conjugated to oligonucleotides for SMA treatment. Among the many hereditary disorders that cause infant mortality, spinal muscular atrophy (SMA) stands out as a motor neuron disease. The present ASO has diminished therapeutic effectiveness due to its restricted distribution in the rostral spinal and brain. Scientists were able to boost levels of survival motor neuron 1 (SMN) protein by synthesizing morpholino oligomer (MO). After injecting MOs-conjugated CPPs such as TAT (YGRKKRRQRRRQ), r6, R6, and (RXRRBR)2XB (RXR) into mice, they measured the resulting SMN protein levels. The results informed their selection of the r6 and RXR peptides, which were then administered to SMA animals exhibiting symptoms. Curiously, during the symptomatic period following intraperitoneal injection, RXR-MO and r6-MO conjugates were detected in the central nervous system, indicating a fully closed blood-brain barrier. The median survival time in symptomatic SMA mice was 41.4 days with RXR-MO conjugates and 23 days with r6-MO conjugates. In the absence of CPP, these values are much more than 17 days for bare MO. According to pathological evidence, CPP-MOs are more successful than scrambled or naked MOs at reversing neuromuscular junction degeneration, proving that CPPs can be used in gene therapy.
Notable CPPs that are found to improve ASO efficiency. (Leckie J., et al., 2024)
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References
- Taylor R E., et al., Cell penetrating peptides, novel vectors for gene therapy, Pharmaceutics, 2020, 12(3): 225.
- Geng J., et al., Emerging landscape of cell-penetrating peptide-mediated nucleic acid delivery and their utility in imaging, gene-editing, and RNA-sequencing, Journal of Controlled Release, 2022, 341: 166-183.
- Leckie J., et al., Potential of Cell-Penetrating Peptide-Conjugated Antisense Oligonucleotides for the Treatment of SMA, Molecules, 2024, 29(11): 2658.
- Nam S H., et al., Recent advances in selective and targeted drug/gene delivery systems using cell-penetrating peptides, Archives of Pharmacal Research, 2023, 46(1): 18-34.
