Journal: bioRxiv

Article Title: Defining a Midgestational Window for In Utero Genome Editing of the Fetal Murine Cortex

doi: 10.64898/2026.04.28.721509

Figure Lengend Snippet: Eight embryos were injected in utero at E15.5 with a pooled barcoded library of seven capsids (AAV9, AAV-DJ, AAV1, AAV6, CAP-B10, PHP.eB, X1.1) at equal concentrations and collected at E18.5. Data were analyzed using linear mixed effects models with tissue, capsid, and normalized reads as fixed effects, and biological replicates as a random effect, with AAV9 set as the reference capsid. A-B) Normalized DNA (A) and RNA (B) abundance of each capsid in cortex, caudal brain, eyes, liver, heart, and lung, relative to AAV9, quantified by NGS. Brain-biased capsids (PHP.eB, CAP-B10, X1.1; tropism established in adult mice) showed no statistically significant enrichment in CNS tissues or depletion in liver (all p > 0.05). C-D) Normalized DNA (C) and RNA (D) abundance in gonads (ovaries and testes; n = 2 female, n = 6 male).

Article Snippet: Self-complementary pAAVs were generated from pscAAV-CAG-GFP, a gift from Mark Kay (Addgene, 83279). pAAV2/1 (gift from James M. Wilson; Addgene plasmid #112862), pAAV2/2 (gift from Melina Fan; Addgene plasmid #104963), AAV3 rep-cap (Cell Biolabs, VPK-423), AAV4 rep-cap (Cell Biolabs, VPK-424), pAAV2/5 (gift from Melina Fan; Addgene plasmid #104964), AAV6 rep-cap (Cell Biolabs, VPK-426), pAAV2/7 (gift from James M. Wilson; Addgene plasmid #112863), pAAV2/8 (gift from James M. Wilson; Addgene plasmid #112864), pUCmini-iCAP-AAV-PHP.eB13 (Addgene #103005), pUCmini-iCAP-AAV.CAP-B10 (Addgene #175004), AAV-DJ rep-cap (Cell Biolabs, VPK-420-DJ), and pUCmini-iCAP-AAV9-X1.1 (Addgene #196836) were used for production of AAVs together with the adenoviral helper plasmid pHelper (Agilent Technologies, #240071).

Techniques: Injection, In Utero

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: iPSC, HSPC and T cells were edited at CCR5 locus using RNP and AAV6 gene editing for knock-in of short sequence (two stop codons) with different concentrations of AZD7648 and TFU72 as indicated. HDR, NHEJ, MMEJ and WT allelic frequencies were calculated at D3 post editing using Sanger Sequencing and ICE analysis (n=3). A, C, E . Scatter plots show the allelic HDR frequency in iPSC ( A ), HSPC ( C ) and T cells ( E ). B, D, F . Bar graphs show the distribution of allelic HDR, NHEJ, MMEJ and WT frequencies in iPSC ( B ), HSPC ( D ) and T cells ( F ). GT denotes gene targeting (HDR) with RNP, AAV6 editing. A and T denote AZD7648 and TFU72, respectively.

Article Snippet: AAV6 vectors were produced in-house or acquired through Vigene, Signagen, or Vectorbuilder.

Techniques: Knock-In, Sequencing

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: iPSC ( A ), HSPC ( B ) and T cells ( C ) were edited individually at CCR5, HBB and STING1 loci with two different gRNAs at each loci using RNP and AAV6 gene editing for the knock-in of short sequence with AZD7648 and TFU72 at the indicated concentrations. Bar graphs show allelic HDR frequencies (% targeted alleles) measured at D3 post editing using Sanger Sequencing and ICE analysis (n=3). GT denotes gene targeting (HDR) with RNP, AAV6 editing. AAV6 HDR template for CCR5 locus was designed to knock-in two stop codons at the target site. At the HBB locus, the AAV6 HDR template was designed to knock-in silent mutations and correction of the sickle cell disease mutation (E6V). For the STING1 locus, the AAV6 HDR template was designed to knock-in a point mutation (V155M) along with silent mutations at the gRNA target site.

Article Snippet: AAV6 vectors were produced in-house or acquired through Vigene, Signagen, or Vectorbuilder.

Techniques: Knock-In, Sequencing, Mutagenesis

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: A-C . iPSC ( A ), HSPC ( B ) and T cells ( C ) were edited at CCR5, HBB and STING1 loci individually using RNP and AAV6 gene editing for the knock-in of multi-kb sequence with AZD7648 and TFU72 at the indicated concentrations and at different AAV6 doses, multiplicity of infection (MOI): 500, 1000, 2500, 5000. Bar graphs show allelic HDR frequencies (% targeted alleles) measured at D3 post editing using ddPCR analysis (n=3). GT denotes gene targeting (HDR) with RNP, AAV6 editing. AAV6 HDR templates for CCR5 and HBB loci were designed to knock-in a 2.2-kb sequence consisting of UBC promoter driven GFP followed by a bGH polyA signal sequence. For the STING1 locus, the AAV6 HDR template was designed to knock-in a 1.4-kb sequence consisting of PGK promoter driven GFP followed by a sv40 polyA signal sequence.

Article Snippet: AAV6 vectors were produced in-house or acquired through Vigene, Signagen, or Vectorbuilder.

Techniques: Knock-In, Sequencing, Infection

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: HSPCs were edited at HBB locus using RNP, AAV6 (Hifi or WT Cas9) with AZD7648 (A, 0.5 µM) or TFU72 (T, 0.5 µM) or no treatment (U). Mock electroporated cells (M) were used as a negative control. GT denotes gene targeting (HDR) with RNP, AAV6. A . At D3 post editing, a previously characterized off-target site at Chr9 (OT1) was PCR amplified from genomic DNA and sequenced by NGS. Bar graphs show INDEL frequency at the OT1 site as determined by the CRISPResso2 tool analysis of the NGS data (n=2). WT Cas9 edited samples were assessed for the INDELs using Sanger Sequencing and ICE analysis. B . Translocation between on-target (HBB) site and OT1 off-target site was assessed by ddPCR analysis and data is shown as percentage of translocation which is the sum of 4 different translocation outcomes (n=2). C . HSPCs edited at HBB loci with Hifi Cas9 were assessed for the frequency of large deletions at the on-target site using Nanopore sequencing of a 10-kb PCR amplicon. Bar graph shows the frequency of reads with deletions of the sizes 50-1000, 1000-3000, 3000-5000 and above 5000 bp (n=3). D-E. HSPCs edited at HBB locus using RNP, AAV6 with different incubation times of AAV6 (U), AZD7648+AAV6 (A) and TFU72+AAV6 (T) were assessed for the off-target INDELs at the OT1 site as described above. D. Bar graphs show the INDEL frequency at the OT1 site at D3 post editing in samples with 12h, 24h and 72h incubation times (n=1). E. Bar graphs show the frequency of reads with deletions of the sizes 50-1000, 1000-3000, 3000-5000 and above 5000 bp as determined by Nanopore sequencing described above (n=1).

Article Snippet: AAV6 vectors were produced in-house or acquired through Vigene, Signagen, or Vectorbuilder.

Techniques: Negative Control, Amplification, Sequencing, Translocation Assay, Nanopore Sequencing, Incubation

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of AAV6-mediated transduction of iMG. Cells were transduced with an MOI of 125000 at the time of plating. On day 3, cells were incubated with 2 μg/mL of pHrodo-labeled oligomerized amyloid (1-42) and imaged over a 20-hour window. (B) Protein lysates isolated from iMG 72 hours after transduction were used to quantify percent CD33 protein expression by MSD. Data are normalized to no AAV6 transduction control. (C) Confocal fluorescence images of iMG assessed for internalization of pHrodo-oligomerized amyloid beta treated as in (A). Digital phase contrast was used to visualize iMG. Images are stitched from four 20X images. Scale bar represents 400 μm. (D) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry per hour. Data are normalized to no AAV6 transduction control. (E) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry at endpoint. Data are normalized to no AAV6 transduction control. (F) TREM2 secretion in conditioned media was quantified from iMG 72 hours after transduction by MSD. (G)(left) Percent change in TREM2 secretion in conditioned media was quantified from HMC3 cells 48 hours after transduction with increasing AAV6-CD33 concentrations by MSD. Data are represented as a percent change relative to dose-matched AAV6-mCherry. (right) TREM2 secretion in conditioned media was quantified from HMC3 cells 48 hours after transduction of AAV6-CD33 by MSD. (MOI = 250000). (H) Protein lysates isolated from HMC3 cells 48 hours after transduction were used to quantify percent full length TREM2 protein expression by MSD. (MOI = 250000). Data represent mean ± SEM using one-way ANOVA with post-hoc Tukey’s multiple comparisons test (B), with uncorrected Fisher’s LSD (E), with post-hoc Dunnett multiple comparisons test (F, G) and one-tailed unpaired t-test (H). Data points (n-numbers) are plotted on each bar graph and scatter plot each representing an independent experimental replicate. Line plots represent three independent experimental replicates (D, G).

Article Snippet: AAV6-mCherry was obtained from Charles River Laboratories.

Techniques: Transduction, Incubation, Labeling, Isolation, Expressing, Control, Fluorescence, One-tailed Test

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of AAV6-mediated transduction of iMG. Cells were transduced with an MOI of 125000 at the time of plating. On day 1, cells were transfected with a di-siRNA targeting CD33 for 48 hours. On day 3, cells were incubated with 2 μg/mL of pHrodo-labeled oligomerized amyloid (1-42) and imaged over a 20-hour window. (B) Confocal fluorescence images of iMG assessed for internalization of pHrodo-oligomerized amyloid beta treated as in (A). Digital phase contrast was used to visualize iMG. Images are stitched from four 20X images. Scale bar represents 400 μm. (C) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry per hour after transfection of increasing concentrations of CD33-targeting di-siRNA per hour. Data are normalized to mock transfected no AAV6 transduction control. (D) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry per hour after transfection of increasing concentrations of CD33-targeting di-siRNA at endpoint. Data are normalized to mock transfected no AAV6 transduction control. (E) Protein lysates isolated from iMG 72 hours after transduction and 48 hours after transfection were used to quantify percent CD33 protein expression by MSD. Data are normalized to mock transfected AAV6-CD33-treated iMG. (F) Linear regression of data from (D) and (E) was plotted to demonstrate the correlation between percent CD33 expression levels and oligomerized amyloid-beta uptake. Data represent mean ± SEM using one-way ANOVA with uncorrected Fisher’s LSD (D) and Dunnett’s multiple comparison test (E). Data points (numbers) are plotted on each bar graph and scatter plot, each representing an independent experimental replicate. Line plots are representative of three independent experimental replicates (C, F).

Article Snippet: AAV6-mCherry was obtained from Charles River Laboratories.

Techniques: Transduction, Transfection, Incubation, Labeling, Fluorescence, Control, Isolation, Expressing, Comparison