HDAd vectors expressing prime editors and in vitro validation. (A) Intended base conversions. The goal is to convert the GTG (Val) codon to a GAA (Glu) codon to repair the sickle mutation (T>A) and to prevent the PE from continuing editing by destroying the PAM (G>A silent mutation). (B) Schematics of HDAd vectors used in this study. The prime editing machinery consists of (1) a prime editing guide RNA (pegRNA), capable of identifying the target nucleotide sequence to be edited and encoding new genetic information that replaces the targeted sequence. The pegRNA consists of an extended single guide RNA (sgRNA) containing a primer binding site (PBS) and a reverse transcriptase (RT) template sequence. During genome editing, the primer binding site allows the 3’-end of the nicked DNA strand to hybridize to the pegRNA, whereas the RT template serves as a template for the synthesis of edited genetic information.⁸ epegRNA indicates the addition of engineered stabilizing structure at 3’-end of pegRNA for extended pegRNA expression and improved editing activity.¹⁰ (2) nCas9-RT. The SpCas9 nickase (nCas9) contains a H840A mutation to inactivate one of its two nuclease domains, thereby disabling its capability of making double-stranded DNA breaks and only allowing single strand DNA nicking. The Cas9 nickase is linked to a M-MLV RT capable of synthesizing DNA from a single-stranded RNA template. The nCas9-RT∗ in PEmax vectors designate optimization in codon usage, nuclear localization signals, and nCas9 activity through mutations. (3) sgRNA-nick is the sgRNA that directs the nCas9 to nick the nonedited DNA strand. The nick location of sgRNA-nick1 is 72 bp away from the pegRNA-induced nick. SgRNA-nick2 spacer partially overlaps with the pegRNA spacer and only matches with the PAM-containing strand after editing occurs, thereby minimizing indel frequency. (4) dominant negative MLH1 inhibits endogenous MLH1 through inhibition, thereby reducing cellular mismatch repair responses and increasing prime editing efficiency.⁹ Additional elements of the PE cassette include U6: U6 RNA polymerase III promoter; EF1α: Elongation factor 1α promoter, miRNA/β-3’UTR: miR-183-5p and miR-218-5p target sites embedded into β-globin to suppress nCas9-RT expression in HDAd producer cells, thus avoiding vector rearrangements, and supporting high vector production yields³⁰; (pA1: BGH pA; pA2: SV40 pA, pA3: rabbit β-globin pA). The vectors also contain a PGK-MGMT^P140K expression cassette used before.¹⁶ Note that PE3 is an earlier version of the prime editing system⁸ whereas PEmax consists of codon- and activity-optimized nCas9-RT components.⁹ (C) Analysis of G>A (silent PAM site) editing in cell lines. Human embryonic kidney (HEK) 293 and erythroid K562 cells (both without the SCD mutation) were transduced with HDAd vectors expressing either PE3, PE3max, PE3bmax, or PE5max at the indicated MOIs. Three days later, DNA was subjected to Sanger sequencing. Data shown here are from 2 independent experiments. (D) Editing of the target T>A site in Lin^– BM cells from SCD CD46/Townes mice. Lin^– cells, a fraction that is enriched for HSPCs, was infected with the 3 HDAd-PE vectors at an MOI of 500 vp/cell and editing was analyzed 4 days later using NGS of the target region. n = 3 donor mice; ∗P < .05. (E) Analysis of G>A (silent PAM site) editing in CD34⁺ cells from 3 healthy donors using NGS. Editing was measured at 4 days after transduction. MOI = 500 vp/cell. ∗P < .05. Statistical significance was assessed using one-way ANOVA with Šidák’s multiple comparisons test to calculate P values. pA, polyadenylation signals; UbC, human ubiquitin C promoter.³¹