Volume 19, Issue 1, Pages 172-180 (January 2011) Nanoparticles Deliver Triplex-forming PNAs for Site-specific Genomic Recombination in CD34+ Human Hematopoietic Progenitors  Nicole A McNeer, Joanna Y Chin, Erica B Schleifman, Rachel J Fields, Peter M Glazer, W Mark Saltzman  Molecular Therapy  Volume 19, Issue 1, Pages 172-180 (January 2011) DOI: 10.1038/mt.2010.200 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Nanoparticle properties. (a) Nanoparticles show uniform size and morphology. Scanning electron microscope images of nanoparticle batches. “Blank” nanoparticles were loaded with phosphate-buffered saline. Average particle diameter and SD given under each batch name. (b) Nanoparticles can be densely loaded with DNA and/or PNA. Batches were loaded with 1 nmole DNA + 13.5 µg spermidine/mg PLGA (“DNA”), 0.5 nmole PNA + 0.5 nmole DNA/mg PLGA (“PNA-DNA”), or 1 nmole PNA/mg PLGA (“PNA”). Loading of PNA and DNA per mg of nanoparticles is given ± SD, n = 4 for each batch. Below the loading data, percent release of nucleic acid after 24 hours incubation at 37 °C is given. PLGA, poly(lactic-co-glycolic acid); PNA, peptide nucleic acid. Molecular Therapy 2011 19, 172-180DOI: (10.1038/mt.2010.200) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Nanoparticle uptake by human CD34+ cells. (a) PLGA nanoparticles readily associate with and are taken up by hematopoietic cells. CD34+ cells were plated overnight, then coumarin 6 loaded nanoparticles (206 ± 73 nm) were added at the indicated concentrations. Uptake was measured by FACS (arbitrary fluorescence units) at day 1 and day 3 of treatment. Trypan blue staining was used to determine external (cell-association) versus internal particles (uptake) because Trypan blue can quench any fluorescence from external particles. (b) Example of histogram showing signal from coumarin 6 at day 1. Using untreated as baseline, 80.9% of cells treated with 0.2 mg/ml coumarin 6 showed internalization, and 99.1% of cells treated with 2 mg/ml showed internalization. Cells are 98% CD34+ at day 1 (see Figure 2). (c) Particle uptake by CD34+ cells was confirmed by confocal microscopy. Cells were stained with Texas Red Phalloidin and DAPI (Blue). Coumarin 6 particles are green. FACS, fluorescence-activated cell sorting; PLGA, poly(lactic-co-glycolic acid). Molecular Therapy 2011 19, 172-180DOI: (10.1038/mt.2010.200) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Cell viability. Data given with SD where appropriate. P values are for two-tailed t-test, 2-sample unequal variance. “Untreated” cells were maintained in regular media without additional manipulation. Particles were added directly to cells. For nucleofection, cells were harvested by centrifugation and nucleofected as per the Amaxa protocol. All treatment groups began with cells from identical populations of CD34+ cells from the same pool. All treatment groups began with an identical number of cells for each experiment. (a) Nucleic acid delivery via nanoparticles shows lower toxicity than delivery via nucleofection. Cell counts were performed using Trypan blue to distinguish between live and dead cells. Counts are normalized to original cell platings. Error bars for live and dead cells give SD where available. **P = 0.01, ***P = 5 × 10−12. (b) CD34 expression of treated cells in nondifferentiating expansion media. Data is given as %CD34+ with SD. Day 1: data for only low dose particle treatments is available. Days 3 and 7: data for all doses available. *P = 0.0002. Molecular Therapy 2011 19, 172-180DOI: (10.1038/mt.2010.200) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Treatment with nanoparticles leads to robust genomic modification in human hematopoietic progenitor cells. (a) Schematic of the PNA-DNA system. The PNA binds within intron 2 of the endogenous β-globin locus. The single-stranded, 50-mer donor DNA molecule is homologous to the β-globin gene, except for a 6-nucleotide sequence change, designed for gene modification at the exon 2/intron 2 boundary that produces a thalassemia-causing mutation. Allele-specific PCR can distinguish between modified (“mutant”) and unmodified (“wild-type”) genomic DNA. (b) Modification is detectable in CD34+-derived cells after 3 days of incubation with nanoparticles loaded with the indicated nucleic acids. Blank = human-CD34+-derived cells treated with nanoparticles loaded with PBS only. Same amount of genomic DNA was used for each PCR. (c) Quantitative PCR of human-CD34+-derived cells at 7 days following nanoparticle treatment shows that the level of genomic modification varies with the nanoparticle dose. Relative levels of modification are given in arbitrary units, with normalization to levels of β-globin wild-type primer amplification. Error bars where indicated give ± SD (n = 3). (d) Genomic DNA, harvested from PNA + DNA nanoparticle-treated CD34+ cells, was gel-purified before allele-specific PCR to exclude theoretical PCR artifact arising from any residual PNA or DNA oligonucleotide. AS-PCR of particle-treated genomic DNA before gel purification is shown for comparison. (e) Genomic DNA from untreated CD34+ cells was spiked with donor DNA oligonucleotide immediately prior to PCR. No PCR amplification using mutant-specific primers is observed when DNA oligonucleotide is added directly to wild-type genomic DNA (“spike” lane). (f) Modification is detectable in differentiated cells, and persists in human-CD34+-derived cells up to 30 days in culture. Low-dose particle treated or nucleofected cells were grown in erythroid- or neutrophil-differentiating conditions, or in media with expansion (nondifferentiating) cytokines (“expansion”), and routinely harvested for detection of presence of the β-globin mutant. (g) Nanoparticles-containing PNAs and donor DNAs targeting CCR5 show applicability of nanoparticles for delivery of gene targeting agents to human CD34+ cells. Genomic DNA harvested from cells 3 days following nanoparticle treatment shows targeted modification at this alternate site. Plasmids containing the mutation or wild-type sequence of the human gene verify specificity of allele-specific primers. Same amount of genomic DNA was used for each PCR. AS-PCR, allele-specific PCR; Blank, CD34 cells treated with particles containing PBS only; PLGA, poly(lactic-co-glycolic acid); PNA, peptide nucleic acid; Untr, untreated CD34+ cells (cells in culture medium only). Molecular Therapy 2011 19, 172-180DOI: (10.1038/mt.2010.200) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Estimation of gene modification frequency in CD34+ cells treated with PNAs and DNAs targeting the β-globin gene, either by amaxa nucleofection or via nanoparticles. (a) Quantitative AS-PCR using primers specific for the introduced mutation was performed on genomic DNA from particle-treated or nucleofected-CD34+ cells, and relative values (normalized to wild-type AS-PCR, n = 3) were compared to a standard curve generated by quantitative AS-PCR with known amounts of mutant plasmid copies. Increasing amounts of pcDNA4 with the mutant human β-globin gene, containing the same modification as that introduced by the donor DNA oligonucleotide, were added to wild-type genomic DNA from untreated CD34+ cells, and subjected to quantitative AS-PCR using primers specific for the targeted modification. The resulting normalized values were plotted against the calculated mutant allele frequency, generating a standard curve that was used to estimate modification frequencies of nanoparticle- and nucleofected-CD34+ samples (depicted by square and triangle symbols, respectively). The circle symbol denotes a PCR sample in which purified wild type genomic DNA was spiked with donor DNA oligonucleotide immediately prior to the PCR. This control PCR was to assure that the presence of single-stranded DNA donor oligonucleotides would not serve as artifact for the mutant AS-PCR. (b) Schematic of the limiting/low dilution assay to independently determine modification frequency. (c) Comparison of estimated modification frequencies calculated using the plasmid standard curve or limiting dilution assay in nanoparticle-treated or nucleofected-CD34+ cells, at the indicated time points following treatment. 95% confidence intervals are given in parentheses. AS-PCR, allele-specific PCR; PLGA, poly(lactic-co-glycolic acid); PNA, peptide nucleic acid. Molecular Therapy 2011 19, 172-180DOI: (10.1038/mt.2010.200) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions