Hydrodynamics-Based Transplacental Delivery as a Useful Noninvasive Tool for Manipulating Fetal Genome

Abstract

We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the EGFP cDNA) complexed with the gene delivery reagent, resulted in some fetuses exhibiting reduced fluorescence in their hearts and gene insertion/deletion (indel) mutations. In this study, we examined whether the endogenous myosin heavy-chain α (MHCα) gene can be successfully genome-edited by this method in the absence of a gene delivery reagent with potential fetal toxicity. For this, we employed a hydrodynamics-based gene delivery (HGD) system with the aim of ensuring fetal gene delivery rates and biosafety. We also investigated which embryonic stages are suitable for the induction of genome editing in fetuses. Of the three pregnant females injected at E9.5, one had mutated fetuses: all examined fetuses carried exogenous plasmid DNA, and four of 10 (40%) exhibited mosaic indel mutations in MHCα. Gene delivery to fetuses at E12.5 and E15.5 did not cause mutations. Thus, the HGD-based transplacental delivery of a genome editing vector may be able to manipulate the fetal genomes of E9.5 fetuses.

Hydrodynamic Delivery: Characteristics, Applications, and Technological Advances

“…Mice [1,2,12] and rats [12,19] are the animals most commonly used for systemic injection from the tail vein; treeshrews are systemically injected using the retro-orbital sinus [20], and chickens using the jugular vein [21]. The liver is the primary organ affected by systemic injection, but substantial transgene expression has also been confirmed in other organs, such as the kidneys [21], brain capillary endothelial cells [22], and even extraordinary tissues, such as subcutaneously implanted colorectal cancer cells [23,24] and fetuses in pregnant animals [25].…”

“…The epoch-making discovery and establishment of the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system are rapidly progressing toward use in in vivo gene editing. Hydrodynamic delivery plays a key role in delivering the components required for in vivo site-directed gene editing, not only for CRISPR-Cas9 [25,95,203,218,219] but also for other systems, such as prime editors [141], split prime editors [140], adenosine deaminase acting on RNA [226], and adenine base editors [139].…”

In vivo Hepatocyte Genome Manipulation via Intravenous Injection of Genome Editing Components

“…Nakamura et al [84] extended TPGD-GEF and showed that HGD of plasmid pCGSap1-MHC (targeted to endogenous myosin heavy chain α (MHCα) gene) in pregnant females on E9.5 resulted in the generation of genome-edited fetuses with an efficiency of 4.16% (Figure 2). Molecular analysis revealed that the mode of genome editing seen in these fetuses was mosaic (i.e., a mixture of genome-edited and non-genome edited cells).…”

“…Hydrodynamics-based gene delivery (HGD)-based transplacental gene delivery for acquiring genome-edited fetuses (TPGD-GEF) using all-in-one vector pCGSap1-MHCα [84]. First, B6C3F1 females were mated to C57BL/6 male mice.…”

“…However, in mice, there are no reports that HGD is harmful to the behavior of treated mice. As for the effects on fetal development, Nakamura et al [84] reported that, in the case of mice used for HGD, there was no appreciable damage to fetuses or the pregnant mother. In this regard, HGD appears to be a safe tool, enabling efficient delivery of NAs to internal organs, including the liver, in mice.…”