CCR5 Gene Disruption by Peptide Nucleic Acids
A new method to permanently alter the sequence of CCR5 in human cells is reported. Transfection of cells produced 2.46% targeted gene modification, which was estimated to be clinically relevant.
Peptide nucleic acids (PNAs) represent a class of synthetic oligonucleotides capable of triple helix formation with high affinity and specificity for duplex DNA.
In this report (1), this technique was used to permanently alter the sequence of CCR5 in human cells and insert a stop codon in the vicinity of the delta 32 mutation site. CD34+ human hematopoietic stem cells were modified in vitro with PNAs and successfully engrafted to NOD-scid IL2rγnull mice.
Four month later, the authors found that multiple lineages of hematopoietic cells, including CD4+ cells, bearing the targeted modification had been produced. The frequency of off-target (CCR2) modification was <0.057%.
According to the authors, this technique is safer than Zinc Finger Nucleases which have an oncogenic risk.
They also have developed (2) a nanoparticles technology to deliver these gene correcting peptides to the cells. This mode of delivery was 60 fold more effective than nucleofection.
Biodegradable nanoparticles can therefore deliver genome-editing agents like PNAs which are able to target and correct disease-causing mutations, and allow a systemic delivery of complex nucleic acid mixtures designed for gene therapy.
Peter M. Glazer (Yale University School of Medicine, New Haven, CT, USA) and Erica Schleifman, co-authors of this paper, kindly agreed to answer our questions.
Alain Lafeuillade: Could you explain us more practically how this gene therapy works and how it is delivered to the cells?
Peter M. Glazer: We use DNA binding molecules called peptide nucleic acids (PNA) that bind sequence specifically to double-stranded DNA. When the PNA is added to cells it finds its target site and binds, forming a very stable PNA/DNA/PNA triple helix. This abnormal structure is recognized by the DNA repair machinery, sensitizing the region of genomic DNA to homologous recombination. If a single-stranded DNA donor oligonucleotide, that is completely homologous to the target site except for your sequence change of interest, is added to the same cells you can selectively induce homologous recombination between the donor and the endogenous gene leading to a precisely prescribed sequence change. his technology can be used to both introduce a mutation, such as the inframe stop codon in the CCR5 gene, or correct a mutation, as we have done in the β-globin locus.
To deliver our molecules we had previously beeen focused solely on electroporation or nucleofection but more recently we have begun utilizing two other approaches. Rogers et al (Molecular Therapy 2011) describes the addition of cell-penetrating peptides to the PNAs for in vivo delivery and increased uptake in cells. We are now currently testing the addition of similar peptides on our donor oligonucleotide for both ex vivo and in vivo studies. Another approach has been the encapsulation of the PNA and donor DNA into polymeric nanoparticles made of the FDA approved polymer, PLGA. In McNeer et al, (Molecular Therapy, 2010) we described the use of these NPs to delivery our molecules into primary CD34+ stem cells with high efficiency and low toxicity. We are currently studying the use of these NPs for both ex vivo and in vivo gene targeting.
AL: Compared with Zinc Finger Nucleases which are currently studied with the same objective of knocking out CCR5 expression to create cells resistant to HIV, what could be the advantages of your technique?
PMG: The advantage that our approach may have over zinc finger nucleases is a much lower risk of off-target mutations in the genome (at least 1000-fold less). Off-target effects of zinc finger nucleases (meaning double strand breaks elsewhere in the genome) could lead to chromosome rearrangements and consequently leukemia or other cancers. Our technology also allows for the introduction of a prescribed sequence change while zinc- finger nuclease-induced double-strand breaks leads to an unpredictable mixture of mutations and chromosomal translocations. PNA-mediated approaches may be advantageous in this regard and may ultimately carry much less risk of untoward side effects in patients. As we report in this paper, when directly compared, the off-target frequency in CCR2 in our PNA-mediated approach, with an upper limit of 0.057%, is at least two orders of magnitude less than that produced by zinc-finger nucleases (Perez et al., 2008).
Finally, unlike with zinc finger nucleases, we do not use viral vectors to deliver our gene targeting molecules as these viral vectors have been known to integrate in the genome inducing oncogene expression.
AL: What are the next steps before moving to clinical trials ?
PMG: Our next steps are to continue to optimize the delivery of our gene targeting molecules with both cell-penetrating peptides and polymeric nanoparticles, as well as, look for new ways to increase our targeting efficiency while retaining our low off-target effects. Preliminary studies in a mouse model using polymeric nanoparticles has been very promising and we hope that further optimization will lead to a gene therapy that can easily move into clinical trials.
1-Schleifman EB, Bindra R, Leif J et al. Targeted Disruption of the CCR5 Gene in Human Hematopoietic Stem Cells Stimulated by Peptide Nucleic Acids. Chem Biol 2011; 18: 1189-98
2-McNeer NA, Chin JY, Schleifman EB, et al. Nanoparticles deliver triplex-forming PNAs for site-specific genomic recombination in CD34+ human hematopoietic progenitors. Mol Ther 2011; 1: 172-80
Key words: HIV cure, HIV functional cure, HIV gene therapy, HIV peptide nucleic acids, zinc finger nucleases