Liquid foam improves potency and safety of gene therapy vectors – Nature Communications

Ethical statement

Our research complies with all relevant ethical regulations: The care and use of mice in this study were approved by the Institutional Animal Care & Use Committee (IACUC) at the Fred Hutchinson Cancer Center and complied with all relevant ethical regulations for animal testing and research (Assurance #A3226–01, IACUC Protocol Number 50782).

Cell lines

HeLa cells for in vitro transfection assays were obtained from ATCC (Cat# CCL-2) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.11 g/liter sodium pyruvate, 2 mM l-glutamine, 4.5 g/liter glucose, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin in a humidified incubator at 5% (v/v) CO₂. Cells tested negative for mycoplasma using a DNA-based PCR test (DDC Medical).

mRNA synthesis

The reporter gene mRNA, CleanCap® F-Luc (Cat# L-7202-5), was purchased from TriLink Biotechnologies (San Diego, CA).

LNP preparation

LNPs were prepared using a previously described method with minor modifications¹⁵. Lipids were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 (SM-102: DSPC: cholesterol: DMG-PEG2000). mRNA was diluted with 6.25 mM sodium acetate buffer (pH 5) to 0.1 mg/mL. mRNA and lipids were combined in a Dolomite micromixer chip at a volume ratio of 3:1 (aqueous:ethanol) and flow rates of 4.5 mL/min (aqueous) and 1.5 mL/min (ethanol). The buffer of the resulting formulation was exchanged for PBS using Amicon Ultra centrifugal filters (100 K NMWL). To prepare DiD’-labeled LNPs, DiD’ (1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt; ThermoFisher Scientific, Cat#: D7757) was incorporated in the initial lipid mixture at a concentration of 0.05 mg/mL. Size distribution was measured via Nanoparticle Tracking Analysis using a NanoSight NS300 (Malvern), and zeta potential was determined using dynamic light scattering detected with a ZetaPALS instrument (Brookhaven). The particles were diluted 1:100 (v/v) in PBS for size measurements, and 1:33 (v/v) in water for zeta potential quantitation.

Lentivirus production

Replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles that constitutively express firefly luciferase under the CMV promoter were purchased from BPS Biosciences (Cat# 79692). Exact titers (transduction units per ml) were provided with each shipment.

Foam preparation

Methylcellulose HV, xanthan gum, and sodium caseinate were obtained from Modernist Pantry.

Albumin from human serum was obtained from Sigma Aldrich (Cat# A1887).

Methylcellulose, sodium caseinate, or albumin (80 mg) and xanthan gum (50 mg) were dissolved in 10 mL PBS to produce the foam precursor. To create foam, 2 mL of the foam precursor was mixed with 18 mL of air via 30 passes between two 20-mL syringes connected at 90° with a 3-way Luer adapter.

Ex vivo transfection screening

Horizontal transfection

HeLa cells were plated into six-well plates and grown to 60-70% confluency. Before transfection, we removed all media from the wells and added 1 mL of fresh DMEM to each well. We then added 3 mL LNP suspension in DMEM or 3 mL foam with embedded LNPs. Two micrograms of firefly luciferase mRNA were added/well (encapsulated in LNPs). Following transfection, cells were incubated at 37 °C for 20 h. We then added 3 mL DMEM to each well, aspirated all foam and media, replaced it with 3 mL of fresh DMEM, and incubated the plates for an additional 25 h before quantitating gene expression using IVIS imaging.

Angled transfection (see also Supplementary Fig. 2)

HeLa cells were plated into six-well plates coated with 2 mL PureCol EZ Gel collagen gel (Sigma-Aldrich) per well and grown to 60–70% confluency. Before transfection, we removed all media from wells and, with plates in a vertical orientation, added 3 mL LNP suspension in PBS or 3 mL foam with embedded LNPs. Two micrograms of firefly luciferase mRNA were added/well (encapsulated in LNPs). We then covered the plates and incubated them at 37 °C at an 120° angle for 2 h. The plates were then returned to a horizontal orientation, and we added 3 mL DMEM to each well, aspirated all foam and media, replaced it with 3 mL of fresh DMEM, and incubated the plates for an additional 25 h before quantitating gene expression using IVIS imaging.

Foam characterization

Freshly prepared foam was characterized by Krűss USA (Matthews, NC, USA) using a Dynamic Foam Analyzer DFA100FSM (Krűss Scientific). Data were acquired and analyzed using ADVANCE software.

Confocal microscopy

To visualize LNPs within foam lamellae (Fig. 3b, c), we freshly prepared methylcellulose/xanthan gum foam with embedded DiD’-labeled liposomes and directly added one drop of foam via an 18-gauge needle onto a 10-mm PTFE printed ring microscopy slide (Delta Microscopies, Cat#: D63417-13) before covering the slide with a 12-mm round coverslip. Fluorescent and DIC (Differential Interference Contrast) images of lamellae, which were defined as channels between two air bubbles in the foam, were collected using an Andor Dragonfly 200 High-Speed Confocal microscope (Oxford Instruments) with a ×63/1.4 (oil) objective. DiD’ was excited with a 637 nm laser and collected with a 698/77 nm Cy5 bandpass emission filter. Images were acquired with an Andor Zyla 4.2 Plus sCMOS camera controlled with Fusion software (version 2.4.0.13). The resulting images were viewed and analyzed using ImageJ (Version 1.53t). For each lamella imaged, a merged image containing both the DIC and fluorescent channels was created, and brightness and contrast were adjusted (identically for all images). A region of interest (ROI) was manually drawn across the lamella between the outer edges of two bubbles using the Straight Line tool. The thickness of this ROI was set to 400 pixels, and the Plot Profile tool was used to quantify the mean gray value along its length (averaged across the 400-pixel width). These data were used to characterize the distribution of LNPs in the lamellae of the foam. Additionally, this distribution was visualized for a given lamella using the Surface Plot tool with the specified ROI to create a 3D plot of the mean gray area as it varied across the length and width of the lamella.

TissueFAXS Imaging of intestinal samples

Mouse intestines were embedded in O.C.T. compound and snap frozen in isopentane (2-methylbutane). Following cryostat sectioning, slides were removed from −80 °C storage, placed on dry ice, and prepared for imaging by applying a drop of ProLong^TM Gold Antifade reagent (Invitrogen) and a coverslip. Fluorescent and transmitted light images of the tissue sections were collected using a TissueFAXS PLUS digital pathology system/slide scanner built on a Zeiss Axio Imager Z2 upright microscope and equipped with a Hamamatsu ORCA-Flash4.0 camera. Using the TissueFAXS Imaging Software (version 7.1), ROIs along the outer edges of intestinal sections were defined for automated, high-resolution imaging with a ×20/0.8 (air) objective. An EXFO X-Cite metal halide lamp excitation source and a Cy5 filter (excitation 590–650 nm, emission 673–762 nm) were used to detect DiD’ signal. The resulting images were viewed and analyzed using ImageJ (version 1.53t). For each tissue section imaged, a merged image containing both the transmitted light and fluorescent channels was created, and brightness and contrast were adjusted (identically for all images). An ROI was manually drawn along the edge of an intestinal sample using the Segmented Line tool with a spline fit applied. The thickness of this ROI was set to 400 pixels, and the Measure tool was used to quantify the mean gray value in the fluorescent channel within the ROI. These data were then used to compare the presence of DiD’ signal in intestinal samples from foam-treated and control mice.

Animal studies

Four- to six-week-old female albino B6 (C57BL/6J-Tyr<c-2J>) mice (Strain #:000058) used in all in vivo experiments were obtained from Jackson Laboratory. For intraperitoneal (i.p.) gene transfer, mice were injected i.p. with 1.5 mL of PBS or freshly prepared foam containing an equal dose of gene therapy vector. In nonviral gene therapy studies, mice were injected with a single dose of LNPs loaded with 1 μg of luciferase-encoding mRNA. In the viral gene therapy studies, we injected a single dose of 5.25 × 10⁶ lentiviral particles.

Flow cytometry

Data were acquired using a BD FACSymphony^TM A5 SE and analyzed with FlowJo v10.8.1. Antibodies and other staining reagents used in flow cytometry are listed in Supplementary Table 1. FACS gating strategies are shown in Supplementary Fig. 3.

ELISA

To quantify lentiviral particles in peritoneal lavage fluid and peripheral blood (Fig. 8C), we used an HIV1 p24 ELISA kit (Abcam, Cat#: ab218268), according to manufacturer’s instructions.

Toxicity analysis

To measure potential in vivo toxicities of gene therapy foam (Fig. 6), we injected mice (10/group) i.p. with 1.5 mL of PBS or methylcellulose/xanthan gum foam. Forty-eight hours after injection, mice were anesthetized and blood was collected by retro-orbital bleed to determine the complete blood counts. Blood was also collected for serum chemistry analyses (performed by Moichor Animal Diagnostics, San Francisco, CA). Animals were then euthanized with CO₂ to retrieve organs, which were washed with deionized water before fixation in 4% paraformaldehyde. The tissues were processed routinely, and sections were stained with hematoxylin and eosin. The specimens were interpreted by board-certified staff pathologists, in a blinded fashion.

In vivo bioluminescence imaging

We used D-Luciferin (Xenogen) in PBS (15 mg/ml) as a substrate for F-luc. Bioluminescence images were collected with a Xenogen IVIS Spectrum Imaging System (Perkin Elmer). Living Image software version 4.7.3 (Perkin Elmer) was used to acquire (and later quantitate) the data 10 min after i.p. injection of D-luciferin into animals anesthetized with 150 mg/kg of 2% isoflurane (Forane, Baxter Healthcare). Acquisition times ranged from 10 sec to 5 min.

Statistics and reproducibility

The statistical significance of observed differences was analyzed using an unpaired, two-tailed Student’s t test. The P values for each measurement are listed in the figures or figure legends. All statistical analyses were performed using GraphPad Prism software version 9.0. To evaluate whether foam-embedded LNPs were homogenously dispersed within the liquid lamellae (Fig. 3b–d), we calculated the deciles of the average particle density for each image (n = 27) by dividing the distance between two air bubbles into ten equal groups. The boxplots of each decile were generated for visualization. To show whether there is any difference in the average LNP density between those decile groups, we conducted an overall test using the nonparametric Friedman test and pairwise comparisons using the Wilcoxon Signed rank test. No statistical method was used to predetermine sample size. No data were excluded from the analyses. To ensure reproducibility, we randomized mice before injection with a gene therapy vector (in suspension or foam). The Investigators were not blinded to allocation during experiments and outcome assessment.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-48753-9