The potential of adoptive cell therapy can be extended when combined with genome editing. However, variation in the quality of the starting material and the different manufacturing steps are associated with production failure and product contamination. Here, we present an automated T cell engineering process to produce off-the-shelf chimeric antigen receptor (CAR) T cells on an extended CliniMACS Prodigy platform containing an in-line electroporation unit. This setup was used to combine lentiviral delivery of a CD19-targeting CAR with transfer of mRNA encoding a TRAC locus-targeting transcription activator-like effector nuclease (TALEN). In three runs at clinical scale, the T cell receptor (TCR) alpha chain encoding TRAC locus was disrupted in >35% of cells with high cell viability (>90%) and no detectable off-target activity. A final negative selection step allowed the generation of TCRα/β-free CAR T cells with >99.5% purity. These CAR T cells proliferated well, maintained a T cell memory phenotype, eliminated CD19-positive tumor cells, and released the expected cytokines when exposed to B cell leukemia cells. In conclusion, we established an automated, good manufacturing practice (GMP)-compliant process that integrates lentiviral transduction with electroporation of TALEN mRNA to produce functional TCRα/β-free CAR19 T cells at clinical scale.

Therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication but fail to eliminate the virus. Although transplantation of allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for a few patients, this approach is not considered a general therapeutic strategy because of potential side effects. Conversely, gene editing to disrupt the C-C chemokine receptor type 5 (CCR5) locus, which encodes the major HIV coreceptor, has shown to confer resistance to CCR5-tropic HIV strains. Here, an engineered transcription activator-like effector nuclease (TALEN) that enables efficient CCR5 editing in hematopoietic cells is presented. After transferring TALEN-encoding mRNA into primary CD4+ T cells, up to 89% of CCR5 alleles are disrupted. Genotyping confirms the genetic stability of the CCR5-edited cells, and genome-wide off-target analyses established the absence of relevant mutagenic events. When challenging the edited T cells with CCR5-tropic HIV, protection in a dose-dependent manner is observed. Functional assessments reveal no significant differences between edited and control cells in terms of proliferation and their ability to secrete cytokines upon exogenous stimuli. In conclusion, a highly active and specific TALEN to disrupt CCR5 is successfully engineered, paving the way for its clinical application in hematopoietic stem cell grafts.

Today’s available therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication and transmission but fail to eliminate the virus. Although transplantation of an allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for three patients, this approach is not considered a general therapeutic strategy because of potential severe side effects. Conversely, genome editing to disrupt the CCR5 locus that encodes the major HIV coreceptor was shown to confer resistance to R5-tropic HIV strains on the cellular level. Here, we present a clinically relevant and highly efficient approach to produce HIV-1 resistant CD4+ T cells. After transferring mRNA coding for CCR5-targeting TALEN into CD4+ T cells by electroporation, up to 89% of CCR5 alleles were disrupted. Genotyping confirmed genetic stability of the edited cell product and off-target analyses established absence of relevant mutagenic events. When challenging these edited T cells with R5-tropic HIV, we observed protection in a dose-dependent manner. Functional assessments revealed no significant differences between edited and control CD4+ T cells in terms of proliferation capacity and their ability to secrete cytokines upon exogenous stimuli. Overall, we successfully engineered HIV-resistant CD4+ T cells under clinically relevant conditions, paving the way for clinical translation.

Targeted modulation of gene expression represents a valuable approach to understand the mechanisms governing gene regulation. In a therapeutic context, it can be exploited to selectively modify the aberrant expression of a disease-causing gene or to provide the target cells with a new function. Here, we have established a novel platform for achieving precision epigenome editing using designer epigenome modifiers (DEMs). DEMs combine in a single molecule a DNA binding domain based on highly specific transcription activator-like effectors (TALEs) and several effector domains capable of inducing DNA methylation and locally altering the chromatin structure to silence target gene expression. We designed DEMs to target two human genes, CCR5 and CXCR4, with the aim of epigenetically silencing their expression in primary human T lymphocytes. We observed robust and sustained target gene silencing associated with reduced chromatin accessibility, increased promoter methylation at the target sites and undetectable changes in global gene expression. Our results demonstrate that DEMs can be successfully used to silence target gene expression in primary human cells with remarkably high specificity, paving the way for the establishment of a potential new class of therapeutics.

Programmable nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, allow for the introduction of precise modifications in the human genome. As a consequence, targeted genome editing has heralded a new area for gene therapy and the treatment of acquired and inherited disorders. Here, we describe the concept of designer nuclease- mediated genome engineering, discuss risks and potential limitations of its usage, highlight some of the achieved milestones, and review the most prominent studies that have made it from the laboratory to the clinic.

The mitochondrial cytochrome c oxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly process, the availability of Cox1 is under control of a regulatory feedback cycle in which translation of COX1 mRNA is stalled when assembly intermediates of Cox1 accumulate through inactivation of the translational activator Mss51. Here we isolate a cytochrome c oxidase assembly intermediate in preparatory scale from coa1Δ mutant cells, using Mss51 as bait. We demonstrate that at this stage of assembly, the complex has not yet incorporated the heme a cofactors. Using quantitative mass spectrometry, we define the protein composition of the assembly intermediate and unexpectedly identify the putative methyltransferase Oms1 as a constituent. Our analyses show that Oms1 participates in cytochrome c oxidase assembly by stabilizing newly synthesized Cox1.

Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two heme bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3-Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b.

Oxidative phosphorylation is a key process of cellular metabolism and takes place within mitochondria. The respiratory chain complexes in the inner membrane connect the transfer of electrons from reducing equivalents to the final electron acceptor oxygen with the establishment of a membrane potential, which is in turn used for ATP synthesis. Respiratory chain complexes thus contain sequentially acting subunits equipped with prosthetic groups that allow the acceptance or donation of electrons. In general, Fe/S clusters, FAD, and different hemes (a-, b-, and c-types) are used during electron transport. Fe/S cluster assembly and incorporation into respiratory chain complexes are partly understood (Lill, 2009), as well as certain aspects of heme attachment in c-type cytochromes (Steiner et al., 1996; Bernard et al., 2003, 2005; Kranz et al., 2009; Corvest et al., 2010). In contrast to c-type cytochromes, aand b-type hemes of cytochrome oxidase (COX) and complex III (bc1 complex) are not covalently linked but rather noncovalently coordinated by conserved residues. The mechanism by which aand b-type hemes are inserted is currently not known (Mick et al., 2011; Kim et al., 2012). Here, we used the central subunit of the bc1 complex, cytochrome b, as a model to understand how heme incorporation into respiratory chain subunits occurs in the mitochondrial inner membrane. Cytochrome b contains two heme bs, one low-potential heme bL located close to the intermembrane space side of the inner membrane and one high-potential heme bH at the opposite side of the membrane (Yun et al., 1991). Together with cytochrome c1 and the Rieske Fe/S protein, cytochrome b participates in the catalytic reactions of the bc1 complex, termed the Q cycle (Mitchell, 1975; Crofts, 2004; Osyczka et al., 2005). The hemes of cytochrome b are coordinated in a four-helix bundle by four conserved histidines, two located in the second transmembrane domain (H82 and H96 in baker’s yeast) and two in the fourth transmembrane helix (H183 and H197 in baker’s yeast; Yun et al., 1991; Hunte et al., 2000). We recently found that cytochrome b assembles through a series of four intermediates into the bc1 complex. Intermediate I, which is composed of cytochrome b, Cbp4, and the evolutionary conserved assembly factors Cbp3–Cbp6 (Tucker et al., 2013), accumulates at steady state in wild-type yeast cells and therefore likely represents a pool of cytochrome b ready to assemble Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two heme bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3–Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b. Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondrial translation

The Oxa1 protein is a well-conserved integral protein of the inner membrane of mitochondria. It mediates the insertion of both mitochondrial- and nuclear-encoded proteins from the matrix into the inner membrane. We investigated the distribution of budding yeast Oxa1 between the two subdomains of the contiguous inner membrane—the cristae membrane (CM) and the inner boundary membrane (IBM)—under different physiological conditions. We found that under fermentable growth conditions, Oxa1 is enriched in the IBM, whereas under nonfermentable (respiratory) growth conditions, it is predominantly localized in the CM. The enrichment of Oxa1 in the CM requires mitochondrial translation; similarly, deletion of the ribosome-binding domain of Oxa1 prevents an enrichment of Oxa1 in the CM. The predominant localization in the IBM under fermentable growth conditions is prevented by inhibiting mitochondrial protein import. Furthermore, overexpression of the nuclear-encoded Oxa1 substrate Mdl1 shifts the distribution of Oxa1 toward the IBM. Apparently, the availability of nuclear- and mitochondrial-encoded substrates influences the inner-membrane distribution of Oxa1. Our findings show that the distribution of Oxa1 within the inner membrane is dynamic and adapts to different physiological needs.

Respiratory chain complexes in mitochondria are assembled from subunits derived from two genetic systems. For example, the bc(1) complex consists of nine nuclear encoded subunits and the mitochondrially encoded subunit cytochrome b. We recently showed that the Cbp3-Cbp6 complex has a dual function for biogenesis of cytochrome b: it is both required for efficient synthesis of cytochrome b and for protection of the newly synthesized protein from proteolysis. Here, we report that Cbp3-Cbp6 also coordinates cytochrome b synthesis with bc(1) complex assembly. We show that newly synthesized cytochrome b assembled through a series of four assembly intermediates. Blocking assembly at early and intermediate steps resulted in sequestration of Cbp3-Cbp6 in a cytochrome b-containing complex, thereby making Cbp3-Cbp6 unavailable for cytochrome b synthesis and thus reducing overall cytochrome b levels. This feedback loop regulates protein synthesis at the inner mitochondrial membrane by directly monitoring the efficiency of bc(1) complex assembly.

Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady‐state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperature-sensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin.

The inner membrane of mitochondria is especially protein-rich. To direct proteins into the inner membrane, translocases mediate transport and membrane insertion of precursor proteins. Although the majority of mitochondrial proteins are imported from the cytoplasm, core subunits of respiratory chain complexes are inserted into the inner membrane from the matrix. Oxa1, a conserved membrane protein, mediates the insertion of mitochondrion-encoded precursors into the inner mitochondrial membrane. The molecular mechanism by which Oxa1 mediates insertion of membrane spans, entailing the translocation of hydrophilic domains across the inner membrane, is still unknown. We investigated if Oxa1 could act as a protein-conducting channel for precursor transport. Using a biophysical approach, we show that Oxa1 can form a pore capable of accommodating a translocating protein segment. After purification and reconstitution, Oxa1 acts as a cation-selective channel that specifically responds to mitochondrial export signals. The aqueous pore formed by Oxa1 displays highly dynamic characteristics with a restriction zone diameter between 0.6 and 2 nm, which would suffice for polypeptide translocation across the membrane. Single channel analyses revealed four discrete channels per active unit, suggesting that the Oxa1 complex forms several cooperative hydrophilic pores in the inner membrane. Hence, Oxa1 behaves as a pore-forming translocase that is regulated in a membrane potential and substrate-dependent manner.

N-terminal signal sequences mediate endoplasmic reticulum (ER) targeting and insertion of nascent secretory and membrane proteins and are, in most cases, cleaved off by signal peptidase. The mouse mammary tumor virus envelope protein and its alternative splice variant Rem have an unusually long signal sequence, which contains a nuclear localization signal. Although the envelope protein is targeted to the ER, inserted, and glycosylated, Rem has been described as a nuclear protein. Rem as well as a truncated version identical to the cleaved signal sequence have been shown to function as nuclear export factors for intron-containing transcripts. Using transiently transfected cells, we found that Rem is targeted to the ER, where the C-terminal portion is translocated and glycosylated. The signal sequence is cleaved off and accumulates in nucleoli. In a cell-free in vitro system, the generation of the Rem signal peptide depends on the presence of microsomal membranes. In vitro and in cells, the signal peptide initially accumulates in the membrane and is subsequently released into the cytosol. This release does not depend on processing by signal peptide peptidase, an intramembrane cleaving protease that can mediate the liberation of signal peptide fragments from the ER membrane. Our study suggests a novel pathway by which a signal peptide can be released from the ER membrane to fulfill a post-targeting function in a different compartment.

Mitochondria are surrounded by a double membrane system that forms four intra-organelle compartments: the outer membrane, inner membrane, intermembrane space, and matrix. Each of the two membranes contains a unique set of proteins defining specific functions of that membrane. The vast majority of mitochondrial proteins including those of the mitochondrial membranes are nuclear encoded and synthesized as precursor proteins in the cytosol. Subsequently, they are targeted to the mitochondria and become sorted to the correct submitochondrial destination. A small portion of the mitochondrial inner membrane proteins is encoded by the mitochondrial genome. These proteins are synthesized on mitochondrial ribosomes and are inserted by dedicated machinery into the inner membrane. This chapter summarizes our current knowledge of the signals that target mitochondrial membrane proteins to their correct intracellular location, and describes the mechanisms by which mitochondrial translocation machineries recognize precursor proteins and mediate their insertion into mitochondrial membranes.