Experimental design

The main goal of our study was to determine if nanoparticles loaded with PNA and donor DNA could be used alleviate signs of anemia after a single-dose in utero administration in a mouse model of human β-thalassemia. The sample sizes of experiments were selected on the basis of previous experience. Data collection was stopped at a priori defined points for all experiments. For all in utero treatment experiments, animals were randomly assigned to IV, IA, or control treatment groups in a blinded manner. For β-thalassemia mouse experiments, end points were selected based on relevant clinical manifestation of disease in the mouse model. All sample measurements were blinded. For the β-thalassemia survival experiment, age-matched untreated mice were randomly assigned at weaning. The number of replicates for each experiment is stated in each figure legend.

Oligonucleotides

Mini-PEG γPNA monomers were prepared from Boc-(2-(2-methoxyethoxy)ethyl)-L-serine as a starting material by a series of multistep synthetic procedures including reduction, mitsunobu reaction, nucleobase (A, C, G and T) conjugation and then ester cleavage31. At each step, the respective product was purified by column chromatography30. PNA oligomers were synthesized on solid support using Boc chemistry31. The oligomers were synthesized on MBHA (4-methylbenzhydrylamine) resin according to standard procedures of Boc chemistry. A kaiser test was performed at each step to measure complete coupling and double coupling was performed if it was required. The oligomers were cleaved from the resin using an m-cresol/thioanisole/TFMSA/TFA (1:1:2:6) cocktail, and the resulting mixtures were precipitated with ethyl ether, purified by reversed-phase high-performance liquid chromatography (acetonitrile:water) and characterized with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer6. The sequence of γPNA used in this study is H-KKK-JTTTJTTTJTJT-OOO-T C T C T T T C T T T C A G G G C A -KKK-NH 2 . Underlined bases indicate γPNA residues; K, lysine; J, pseudoisocytosine; O, 8-amino-2,6,10-trioxaoctanoic acid linkers connecting the Hoogsteen and Watson–Crick domains of the tcPNA. The single-stranded donor DNA oligomer was prepared by standard DNA synthesis except for the inclusion of three phosphorothioate internucleoside linkages at each end to protect against nuclease degradation (Midland Certified Reagent Company; Midland, TX). The 60 bp donor DNA matches positions 624–684 in β-globin intron 2, with the correcting IVS2-654 nucleotide underlined: 5′-AAAGAATAACAGTGATAATTTCTGGGTTAAGG C AATAGCAATATCTCTGCATATAAATAT-3′.

PLGA nanoparticle synthesis and characterization

PLGA (50:50 ester-terminated, 0.55–0.75 g dl−1, LACTEL absorbable polymers; Birmingham, AL) NPs containing C6 (Sigma; St Louis, MO) or DiD (Thermo Scientific; Rockford, IL) were synthesized using a single-emulsion solvent evaporation technique17. C6 or DiD was added to the polymer solution at a 0.2% wt:wt dye:polymer ratio. PNA/DNA and blank PLGA NPs were synthesized using a double-emulsion solvent evaporation technique modified to encapsulate PNA and DNA oligomers6,65. PNAs and donor DNAs were dissolved in 60.8 μl DNAse-free water. All nanoparticle batches had 2 nmole mg−1 of γPNA and 1 nmole mg−1 of donor DNA. The encapsulant was added dropwise to a polymer solution containing 80 mg 50:50 ester-terminated PLGA dissolved in dichloromethane (800 μl), then ultrasonicated (3 × 10 s) to formulate the first emulsion. To form the second emulsion, the first emulsion was added slowly dropwise to 1.6 ml of 5% aqueous polyvinyl alcohol and then ultrasonicated (3 × 10 s). This mixture was finally poured into 20 ml of 0.3% aqueous polyvinyl alcohol and stirred for 3 h at room temperature. Nanoparticles were then thoroughly washed with 20 ml water (3×) and further collected each time by centrifugation (25,644 × g for 10 min at 4 °C). Nanoparticles were resuspended in water, frozen at −80 °C, and then lyophilized. Nanoparticles were stored at −20 °C after lyophilization6. Blank NPs were loaded with 1 × phosphate-buffered saline and formulated using the double emulsion method described above. Scanning electron microscopy (SEM) was performed using an XL-30 scanning electron microscope (FEI; Hillsboro, Oregon) as previously described17. Dynamic light scattering (DLS) was performed to measure the NPs size (hydrodynamic diameter) and surface charge (zeta potential) using a Malvern Nano-ZS (Malvern Instruments, UK). Nucleic acid release was analyzed by incubating particles (2 mg) in 600 μl 1× phosphate-buffered saline in a 37 °C shaker, spinning down and removing supernatant. The nucleic acid content of the supernatant was measured as the absorbance at 260 nm at the indicated time points.

Mouse models and genotyping

All animal use was in accordance with the guidelines of the Animal Care and Use Committee (IACUC) of Yale University and conformed to the recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996). Animal protocols were approved by the IACUC of Yale University. C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). The IVS2-654 β-thalassemia mice were obtained from Ryszard Kole, University of North Carolina (Chapel Hill, NC)38. Litters from this mouse model were genotyped prior to weaning. Genomic DNA (gDNA) was isolated from tail clippings using the Wizard SV DNA Purification System (Promega; Madison, WI). Genotyping PCR was performed to detect the presence of the human β-globin gene, indicating the mouse has a Hbbth-4/Hbb+ genotype, using a species independent forward primer (complementary to both mouse and human β-globin sequences) and two species dependent reverse primers. Genotyping primers are as follows: forward—5′-CCCTGGGCAGGTTGGTATC-3′; human reverse—5′-AACGATCCTGAGACTTCCACA-3′; and mouse reverse 5′–AGCAGAGGCAGAGGATAGGTC–3′. PCR was performed using high fidelity Platinum TAQ polymerase (Invitrogen; Carlsbad, CA); reaction conditions are as follows: 5.0 μl 10 × HiFi buffer, 3.0 μl MgCl 2 , 1.0 μl dNTPs, 2.0 μl 10 μM forward primer, 1.0 μl 10 μM human reverse primer, 1.0 μl 10 μM mouse reverse primer, 0.8 μl HiFi Taq, 90–400 ng gDNA and remaining volume to 50 μl with dH 2 O. Thermocycler conditions were as follows: 94 °C 2 min, [94 °C 30 s, 55 °C 45 s, 68 °C 1 min] × 35 cycles, 68 °C 1 min, hold at 4 °C. PCR products were run on a 2% agarose gel. The amplicon derived from the mouse reverse primer is 196 bp and the amplicon from the human reverse primer is 508 bp. The presence of bands at both 196 and 508 bp indicates the Hbbth-4/Hbb+ genotype. A single band at 196 bp indicates the Hbb+/Hbb+ genotype. Only heterozygous mice were included in this study.

In utero NP delivery and imaging

Time dated pregnant mice (8–12 weeks old) between 15–18 days post conception were anesthetized with inhaled isoflurane (3% vol/vol for induction, 2% vol/vol for maintenance). The gravid uterus was exposed through a midline laparotomy incision. For the biodistribution studies, lyophilized fluorescent nanoparticles were resuspended by vortex and water bath sonication in 1× dPBS to a concentration of 9 mg ml−1. Intravascular injections were performed at E15.5 and E16.5. A volume of 15 μl of 9 mg ml−1 NP suspension was drawn up into a glass micropipette (tip diameter ~60 μm) and injected intravascularly via vitelline vein of each fetus using a pneumatic microinjector (Narishige; Japan). Intra-amniotic injections were performed at E15.5, E16.5, E17.5 and E18.5. A volume of 20 μl of 9 mg ml−1 NP suspension was injected directly into the amniotic cavity of each fetus. As a positive control to detect NPs in maternal circulation, 100 μl of 9 mg ml−1 C6 NPs were injected intravenously into a mouse pregnant with fetuses at E15.5. Pregnant mice were killed 3 h post DiD PLGA NP injection and fluorescence and x-ray imaging was performed on a Carestream In-Vivo MS FX PRO (Bruker; Billerica, MA). Pregnant mice were also killed 3 h post C6 PLGA NP injection. Fetuses were delivered via cesarean section and washed in PBS. Ex vivo fetal fluorescence stereomicrope imaging was performed on a Leica M80 stereomicroscope (Wetzlar, Germany). Fetuses and maternal organs were fixed overnight in 4% paraformaldehyde (Electron Microscopy Sciences; Hartfield, PA) at 4 °C. The tissues were next dehydrated in 20% sucrose and embedded in Optimal Cutting Temperature (OCT) Compound (Torrance, CA). Frozen 15 μm-thick fetal and maternal liver sections were mounted on glass slides and stained with Hoescht dye. Confocal imaging of the frozen sections was performed on a Zeiss Axio Observer Z1 microscope (Oberkochen, Germany).

For the safety studies, the fetuses of time dated pregnant C57BL/6 females (8–10 weeks old) were injected intravenously at E15.5 as described above with 15 μl of 9 mg ml−1 PNA/DNA NPs. Intra-amniotic injections were performed at E16.5; 20 μl of 9 mg ml−1 blank NPs or PNA/DNA NPs were injected into the amniotic cavity. Untreated pregnant mice were anesthetized and the gravid uterus was exposed as described above. The fetuses were counted, the uterus was returned to the abdomen, and the midline incision was closed. The number of untreated, intravenously and intra-amniotically treated pups surviving was counted at the time of weaning, 21 days. The weight of injected and untreated control pups was measured for a period of 10 months.

The fetuses of time dated pregnant Hbbth-4/Hbb+ mice (mated with Hbbth-4/Hbb+ males) were injected intravenously as described above with 15 μl of either 9 mg ml−1 or 12 mg ml−1 PNA/DNA NPs, correlating to doses of 300 mg kg−1 or 400 mg kg−1, respectively.

Cytokine array analysis

The fetuses of time dated pregnant C57BL/6 females (8–10 weeks old) were injected intravenously at E15.5 as described above with 15 μl of blank NPs (9 mg ml−1), PNA/DNA NPs (9 mg ml−1), or 1× dPBS. After 48 h, fetal plasma samples were collected. Plasma samples from fetuses receiving PBS, blank NPs, or PNA/DNA NPs and untreated fetuses were submitted to the CytoPlex Core Facility at Yale University. The facility performed luminex based cytokine detection and quantification using the Bio-Plex Pro Mouse Cytokine 23-Plex assay available from Bio-Rad (Hercules, CA).

Peripheral blood analysis

Mice were anesthetized using open-drop 30% w/v isoflurane in propylene glycol. A volume of 50–100 μl of blood was collected retro-orbitally using heparinized micro-hematocrit capillary tubes (Fisher Scientific; Pittsburgh, PA) and evacuated into heparinized coated tubes containing 5 μl 0.5 M EDTA acid. Complete blood counts were performed using a Hemavet 950FS (Drew Scientific; Oxford, CT) according to the manufacturer’s protocol. A volume of 1–3 μl of fresh blood was smeared onto glass slides and stained with Wright–Giemsa stain (Sigma-Aldrich; St. Louis, MO) for 20 s. Slides were washed in 1× dPBS for 10 min and air-dried. An additional 10 μl of blood was incubated with 3 μl new methlylene blue reticulocyte stain (Sigma-Aldrich; St. Louis, MO) for 10 min after which blood smears were prepared. A cover slip was mounted on air-dried smears with Cytoseal 60 (Thermo Scientific; Rockford, IL). All blood smears were imaged on an Olympus FSX100 microscope. Two individuals independently counted the number of reticulocytes present in 500 cells. The relative reticulocyte count was calculated as the number reticulocytes in 1000 RBCs divided by ten.

Histology

Spleen images were taken and weights were recorded for Hbbth-4/Hbb+ mice 15–30 weeks post PNA/DNA NP delivery, age-matched untreated Hbbth-4/Hbb+ mice, and wild-type mice. The collected spleens were fixed in 10% neutral buffered formalin and processed by Yale Pathology Tissue Services for H&E staining as well as E-cadherin, CD44, CD71 and CD61 immunohistochemistry. All antibodies were used at a dilution of 1:1000. Spleen sections were imaged on an Olympus FSX100 microscope.

Fetal bone marrow collection and cell sorting

The bone marrow of untreated and PNA/DNA NP treated Hbbth-4/Hbb+ mice were collected 3 days post NP delivery on E18.5 as previously reported36. Freshly isolated fetal bone marrow cells were suspended in ice-cold DMEM + (Dulbecco’s modified Eagle’s medium, 10 mM HEPES, 2% fetal bovine serum [FBS]) at 107 cells ml−1 and stained for 15 min on ice with anti-cKit-PE (eBioscience, Cat #12-1171-82), anti-Sca1-(PerCP)-Cy5.5 (eBioscience, Cat #45-5981-82), and FITC-conjugated lineage marker antibodies [CD4 (eBioscience, Cat #11-0041-82), CD8 (eBioscience, Cat #11-0081-82), Ter119 (eBioscience, Cat #11-5921-82), Gr-1 (eBioscience, Cat #11-5931-82), and CD45 (eBioscience, Cat #11-0452-82)] (Thermo Scientific; Rockford, IL). All antibodies were used at a dilution of 1:100. Samples were then washed with 10 × volume of HBSS + (Hank’s balanced salt solution, 10 mM HEPES, 2% FBS) and centrifuged at 0.4 × g for 8 min at 4 °C. Cell pellets were resuspended in DMEM + and samples were immediately sorted by flow cytometry (BD FACSAria).

Deep sequencing analysis

Genomic DNA (gDNA) from the bone marrow of adult PNA/DNA NP treated Hbbth-4/Hbb+ mice and age-matched untreated Hbbth-4/Hbb+ mice was collected using the Wizard SV DNA Purification System (Promega, Madison, WI) according to manufacturer’s instructions. gDNA was collected from sorted fetal bone marrow cells using a phenol-chloroform extraction method. Cells were digested overnight in 10 mM Tris-HCl (pH 8), 150 mM NaCl, 20 mM ethylenediamine tetracetic acid and 1% sodium dodecyl sulfate, with proteinase K. Digests were subjected to extraction with phenol/chloroform/isoamyl alcohol followed by re-extraction with choloroform, precipitated with KOAc in EtOH, spun down and dried at room temperature and resuspended in dH 2 O. PCR reactions were performed with high fidelity TAQ polymerase (Invitrogen; Carlsbad, CA). Each PCR tube consisted of 28.2 µL dH 2 O, 5 µL 10 × HiFi Buffer, 3 µL 50 mM MgCl 2 , 1 µL DNTP, 1 µL each of forward and reverse primer, 0.8 µL High Fidelity Platinum Taq Polymerase and 10 µL 40 ng/ml gDNA. Thermocycler conditions were as follows: 94 °C 2 min (94 °C 30 s, 55 °C 45 s, 68 °C 1 min) x35 cycles, 68 °C 1 min, hold at 4 °C. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen; Hilden, Germany). PCR products were prepared by end-repair and adapter ligation according to Illumina protocols (San Diego, CA), and samples were sequenced by the Illumina HiSeq 2500 with 75 paired-end reads at the Yale Center for Genome Analysis. Samples were analyzed as previously described5. Briefly, paired-end reads were merged using PEAR (v. 0.9.6)66. The merged reads were mapped to the loci of interest using BWA-MEM aligner (v. 0.7.12)67. The nucleotide composition for each position in the alignment was obtained with in-house python scripts. To study off-target effects, we looked for the presence of 16 bp k-mers of the donor sequence in the off-target sequencing libraries allowing for one mismatch using BBTools. The primers used for β−globin intron 2 were as follows:6 forward primer: 5′-TATCATGCCTCTTTGCACCA-3′; reverse primer: 5′-AGCAATATGAAACCTCTTACATCA-3′. Primers for off-target sites of partial homology were as follows;6 forward primer is listed first: Vascular cell adhesion protein precursor 1 (5′-AGATAATTATTGCCTCCCACTGC-3′ and 5′-AATGGAAGGGCATGCAGTCA-3′); Polypyrimidine tract binding protein (5′-CCCAATCCTGAATCCTGGCT-3′ and 5′-CATACTGATGTCTGTGGCTTGA-3′); Protocadherin fat 4 precursor (5′-AAGCTCAAACCTACCAGACCA-3′ and 5′-AGCTGGAAGCTTCTTCAGTCA-3′); Olfactory receptor 266 (5′-CCCTCTGTGGACTGAGGAAG-3′ and 5′-TGATGAGCTACGGGTATGTGA-3′); Syntaxin-binding protein (5′-CAAAAAGCCTTAAGCAAACACTC-3′ and 5′-TCTCTCCCTCAGCATCTATTCC-3′); Muscleblind-like protein (5′-TGTGTTTGTTTATGGATACTTGAGC-3′ and 5′-GCATGCACAATAAAGGCACT-3′); Ceruloplasmin isoform (5′-CATGGGAAACAGTCAAAAGAAA-3′ and 5′-GTAGGTTTCCCCACAGCTT-3′).

Droplet digital PCR quantification of editing in bone marrow cells and HPCs

Bone marrow was collected from the femurs of Hbbth-4/Hbb+ mice 15 weeks after NP treatment and age-matched untreated Hbbth-4/Hbb+ mice. Hematopoietic progenitor cells (CD3e-, CD11b−, CD19−, CD45R−, Gr-1-, Ter119−) were isolated by magnetic separation using an EasySepTM Mouse Hematopoietic Progenitor Cell Isolation Kit (STEMCELLTM Technologies, Vancouver, CA) according to manufacturer’s instructions. gDNA was extracted from total bone marrow cells or isolated progenitor populations using the Wizard SV DNA Purification System (Promega, Madison, WI) according to manufacturer’s instructions. The concentration of extracted gDNA samples was measured using a QuBit® dsDNA BR assay kit (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Up to 80 ng of gDNA was used for each sample per reaction. PCR reactions were set up as followed: 11 μl 2 × ddPCR™ supermix for probes (no dUTP) (Bio-Rad, Hercules, CA), 0.2 μl forward primer (100 μM), 0.2 μl reverse primer (100 μM), 0.053 μl β-thal probe (100 μM), 0.053 μl wild-type probe (100 μM) (Integrated DNA Technologies, Coralville, IA), 0.5 μl EcoR1, 10 μl gDNA and dH 2 O. Droplets were generated using the Automated Droplet Generator (AutoDG™) (Bio-Rad). Thermocycling conditions were as follows: 95 °C 10 min, (94 °C 30 s, 55.3 °C 1 min – ramp 2 °C/s) x 40 cycles, 98 °C 10 min, hold at 4 °C. Droplets were allowed to rest at 4 °C for at least 30 min after cycling and were then read using the QX200™ Droplet Reader (Bio-Rad). Data were analyzed using QuantaSoft™ software. Data are represented as the fractional abundance of the wild-type allele. The primers used for ddPCR were as follows: forward: 5′-ACCATTCTAAAGAATAACAGTGA-3′, reverse: 5′-CCTCTTACATCAGTTACAATTT-3′. The probes used for ddPCR were as follows: wild-type (FAM): 5′-TGGGTTAAGGCAATAGCAA-3′, β-thal (HEX): 5′-TCTGGGTTAAGGTAATAGCAAT-3′.

Statistical analysis

The data are means ± s.e.m. unless otherwise noted and compared using one-way or two-way ANOVA with repeated measures when appropriate. Bonferroni correction was used to correct for multiple comparisons. Survival data were analyzed using a Log-rank test. Statistical analyses were carried out using GraphPad Prism. A P value of less than 0.05 was considered statistically significant.

Data availability

Additional data and movies are available in the supplementary material. Deep sequencing data has been deposited in the NCBI Sequence Read Archive under accession number SRP142526.