Chloroplast transformation for bioencapsulation and oral delivery using the immunoglobulin G fragment crystallizable (Fc) domain.

Proinsulin Like Growth Factor I (prolGF-I) and myostatin (Mstn) regulate muscle regeneration and mass when intravenously delivered. We tested if chloroplast bioencapsulated forms of these proteins may serve as a non-invasive means of drug delivery through the digestive system. We created tobacco (Nicotiana tabacum) plants carrying GFP-Fc1, proIGF-I-Fc1, and Mstn-Fc1 fusion genes, in which fusion with the immunoglobulin G Fc domain improved both protein stability and absorption in the small intestine. No transplastomic plants were obtained with the Mstn-Fc1 gene, suggesting that the protein is toxic to plant cells. proIGF-I-Fc1 protein levels were too low to enable in vivo testing. However, GFP-Fc1 accumulated at a high level, enabling evaluation of chloroplast-made Fc fusion proteins for oral delivery. Tobacco leaves were lyophilized for testing in a mouse system. We report that the orally administered GFP-Fc1 fusion protein (5.45 µg/g GFP-Fc1) has been taken up by the intestinal epithelium cells, evidenced by confocal microscopy. GFP-Fc1 subsequently entered the circulation where it was detected by ELISA. Data reported here confirm that chloroplast expression and oral administration of lyophilized leaves is a potential delivery system of therapeutic proteins fused with Fc1, with the advantage that the proteins may be stored at room temperature.

Chloroplast transformation for bioencapsulation and oral delivery using the immunoglobulin G fragment crystallizable (Fc) domain.

Proinsulin Like Growth Factor (prolGF1) and myostatin (Mstn) regulate muscle regeneration when intravenously delivered. We set out to test if chloroplast bioencapsulated forms of these proteins may serve as a non-invasive means of drug delivery through the digestive system. We created tobacco (Nicotiana tabacum) plants carrying GFP-Fc1, proIGF-I-Fc1, and Mstn-Fc1 fusion genes, in which fusion with the immunoglobulin G Fc domain improved both protein stability and absorption in the small intestine. No transplastomic plants were obtained with the Mstn-Fc1 gene, suggesting that the protein is toxic to plant cells. proIGF-I-Fc1 protein levels were too law to enable in vivo testing. However, GFP-Fc1 accumulated at a high level, enabling evaluation of chloroplast-made Fc fusion proteins for oral delivery. Tobacco leaves were lyophilized for testing in a mouse system. We report that the orally administered GFP-Fc fusion protein (5.45 µg/g GFP-Fc) has been taken up by the intestinal epithelium cells, evidenced by confocal microscopy. GFP-Fc subsequently entered the circulation where it was detected by ELISA. Data reported here confirm that chloroplast expression and oral administration of lyophilized leaves is a potential delivery system of therapeutic proteins fused with Fc, with the advantage that the proteins may be stored at room temperature.

Multiple sgRNAs for one-step inactivation of the duplicated acetyl-coenzyme A carboxylase 2 (ACC2) genes in Brassica napus.

Efficient plastid transformation in Arabidopsis (Arabidopsis thaliana) requires genetic lines that are hypersensitive to spectinomycin due to the absence of a chloroplast acetyl-coenzyme A carboxylase (ACCase) encoded in the acetyl-coenzyme A carboxylase 2 (ACC2) nuclear gene. To obtain plastid transformation-competent oilseed rape (Brassica napus), we inactivated all nuclear encoded, chloroplast targeted ACCase copies using CRISPR-Cas9. Brassica napus (2n = 38, AACC) is a recent interspecific hybrid of Brassica rapa (2n = 20, AA) and B. oleracea (2n = 18, CC) and is expected to have at least two ACC2 copies, one from each parent. The sequenced genome has two ACC2 copies, one that is B. rapa-like and one that is B. oleracea-like. We designed single-guide RNAs (sgRNAs) that could simultaneously inactivate both nuclear ACC2 copies. We expressed Cas9 from a chimeric egg cell promoter 1.2 (EC1.2p) known to yield homozygous or biallelic mutants in Arabidopsis in the T1 generation. To maximize the probability of functionally inactivating both orthologs in a single step, each of the two vectors carried four sgRNAs. Four T0 transgenic lines were obtained by Agrobacterium tumefaciens-mediated hypocotyl transformation. Amplicon sequencing confirmed mutations in ACC2 genes in 10 T1 progeny, in seven of which no wild-type (WT) copy remained. The B. napus T2 seedlings lacking WT ACC2 gene copies exhibited a spectinomycin hypersensitive phenotype, suggesting that they will be a useful resource for chloroplast genome transformation.

Rapid sequence evolution is associated with genetic incompatibilities in the plastid Clp complex.

KEY MESSAGE: Replacing the native clpP1 gene in the Nicotiana plastid genome with homologs from different donor species showed that the extent of genetic incompatibilities depended on the rate of sequence evolution. The plastid caseinolytic protease (Clp) complex plays essential roles in maintaining protein homeostasis and comprises both plastid-encoded and nuclear-encoded subunits. Despite the Clp complex being retained across green plants with highly conserved protein sequences in most species, examples of extremely accelerated amino acid substitution rates have been identified in numerous angiosperms. The causes of these accelerations have been the subject of extensive speculation but still remain unclear. To distinguish among prevailing hypotheses and begin to understand the functional consequences of rapid sequence divergence in Clp subunits, we used plastome transformation to replace the native clpP1 gene in tobacco (Nicotiana tabacum) with counterparts from another angiosperm genus (Silene) that exhibits a wide range in rates of Clp protein sequence evolution. We found that antibiotic-mediated selection could drive a transgenic clpP1 replacement from a slowly evolving donor species (S. latifolia) to homoplasmy but that clpP1 copies from Silene species with accelerated evolutionary rates remained heteroplasmic, meaning that they could not functionally replace the essential tobacco clpP1 gene. These results suggest that observed cases of rapid Clp sequence evolution are a source of epistatic incompatibilities that must be ameliorated by coevolutionary responses between plastid and nuclear subunits.