In vivo HSC prime editing rescues Sickle Cell Disease in a mouse model

Abstract

Sickle Cell Disease (SCD) is a monogenic disease caused by a nucleotide mutation in the β-globin gene. Current gene therapy studies are mainly focused on lentivirus vector-mediated gene addition or CRISPR/Cas9-mediated fetal globin reactivation, leaving the root cause unfixed. We developed a vectorized prime editing system that can directly repair the SCD mutation in hematopoietic stem cells (HSCs) in vivo in a SCD mouse model (CD46/Townes mice). Our approach involved a single intravenous injection of a non-integrating, prime editor-expressing virus vector into mobilized CD46/Townes mice and low-dose drug selection in vivo. This procedure resulted in the correction of ~40% of bS alleles in HSCs. On average 43% of HbS was replaced by HbA thereby greatly mitigating the SCD phenotypes. Transplantation in secondary recipients demonstrated that long-term repopulating HSCs were edited. Highly efficient target site editing was achieved with minimal generation of insertions and deletions and no detectable off-target editing. Because of its simplicity and portability, our in vivo prime editing approach has the potential for application in resource-poor countries where SCD is prevalent.

Hematopoietic stem cell collection for sickle cell disease gene therapy

“…A complementary strategy may be to reduce the required yields by utilizing emerging methods for ex vivo HSC expansion during DP manufacturing [102,103,104 & ,105]. In the future, efforts to optimize HSC mobilization are likely to facilitate "in vivo" gene therapies by overcoming the challenges of targeting HSCs within the BM space [94,[106][107][108][109]. Presently, improvements in HSC collection are required to improve the safety, efficacy, cost, and availability of current gene therapies for SCD.…”

Prime editing in hematopoietic stem cells—From ex vivo to in vivo CRISPR-based treatment of blood disorders

“…Notably, data disclosed in relation to the initial public offering of Prime Medicine (https://primemedicine.com) suggest that ex vivo prime editing of human CD34 + HSPCs can be quite effective and may support treatment of certain blood disorders. Indeed, a very recent study showed that human adenoviral vector-mediated delivery of prime editors to HSCs allows potent prime editing both ex vivo and in vivo, thereby providing the first proof-of-principle that prime editing of HSCs has the potential to be used as a treatment for blood disorders (Li et al, 2023). Still, progress in the field has been relatively slow, which may reflect different aspects of the technology.…”

“…Recently, a similar approach was used by Li and coworkers to deliver the most optimized prime editing system, referred to as PE5max, to human HSCs ex vivo. By utilizing helper-dependent adenovirus (HDAd5/35++), the authors were able to achieve 3.4% prime editing of a SCD-causing HBB-variant in CD34 + HSPCs from healthy donors and 4.6% in CD34 + cells derived from SCD-patients (Li et al, 2023). Importantly, the authors were able to achieve up to 40% prime editing in vivo in HSCs in a SCD-mouse model (CD46/ Townes) by first mobilizing HSCs to the peripheral blood using G-CSF followed by a single intravenous administration of HDAd5/ 35++ vectors.…”

“…Previous work using conventional gene therapies has circumvented this restriction by either directly injecting lentiviral vectors into the bone marrow (Pan et al, 2004;Worsham et al, 2006) or alternatively by mobilizing HSPCs to the peripheral blood followed by intravenous injection of viral vectors (Richter et al, 2016;Humbert et al, 2018; (Figure 2B). The latter approach was used by Li and coworkers to deliver the PE5max system to HSCs in vivo (Li et al, 2023). For conventional gene therapies, off-target cell transduction remains a concern of these approaches.…”

Nucleotide metabolism constrains prime editing in hematopoietic stem and progenitor cells

“…Combining modulation of nucleotide metabolism, PEmax editors, and MMR evasion designs allows unprecedented prime editing efficiency and purity in HSPCs. Considering recent progress towards delivery of genome editors to HSCs in vivo 25,26 , these findings may help designing novel prime editing therapies to engineer quiescent HSCs in situ .…”