Replacement Vector Research Articles

Versatile allelic replacement and self-excising integrative vectors for plasmid genome mutation and complementation.

In spite of the dissemination of multidrug-resistant plasmids among Gram-negative pathogens, including those carrying virulence genes, vector tools for studying plasmid-born genes are lacking. The allelic replacement vectors can be used to generate plasmid or chromosomal mutations including markless point mutations. This is the first report describing a self-excising integrative vector that can be used as a stable single-copy complementing tool to study medically important pathogens including in vivo studies without the need for antibiotic selection. Overall, our newly developed vectors can be applied for the assessment of the function of plasmid-encoded genes by specifically creating mutations, moving large operons between plasmids and to/from the chromosome, and complementing phenotypes associated with gene mutation. Furthermore, the vectors express chromophores for the detection of target gene modification or colony isolation, avoiding time-consuming screening procedures.

Rescue of GM3 synthase deficiency by spatially controlled, rAAV-mediated ST3GAL5 delivery.

GM3 synthase deficiency (GM3SD) is an infantile-onset epileptic encephalopathy syndrome caused by biallelic loss-of-function mutations in ST3GAL5. Loss of ST3GAL5 activity in humans results in systemic ganglioside deficiency and severe neurological impairment. No disease-modifying treatment is currently available. Certain recombinant adeno-associated viruses (rAAVs) can cross the blood-brain barrier to induce widespread, long-term gene expression in the CNS and represent a promising therapeutic strategy. Here, we show that a first-generation rAAV-ST3GAL5 replacement vector using a ubiquitous promoter restored tissue ST3GAL5 expression and normalized cerebral gangliosides in patient-derived induced pluripotent stem cell neurons and brain tissue from St3gal5-KO mice but caused fatal hepatotoxicity when administered systemically. In contrast, a second-generation vector optimized for CNS-restricted ST3GAL5 expression, administered by either the intracerebroventricular or i.v. route at P1, allowed for safe and effective rescue of lethality and behavior impairment in symptomatic GM3SD mice up to a year. These results support further clinical development of ST3GAL5 gene therapy.

Binocular benefit following monocular subretinal AAV injection in a mouse model of autosomal dominant retinitis pigmentosa (adRP)

Autosomal dominant retinitis pigmentosa (adRP) is frequently caused by mutations in RHO, the gene for rhodopsin. In previous experiments in dogs with the T4R mutation in RHO, an AAV2/5 vector expressing an shRNA directed to human and dog RHO mRNA and an shRNA-resistant human RHO cDNA (AAV-RHO820-shRNA820) prevented retinal degeneration for more than eight months following injection. It is crucial, however, to determine if this RNA replacement vector acts in a mutation-independent and species-independent manner. We, therefore, injected mice transgenic for human P23H RHO with this vector unilaterally at postnatal day 30. We monitored their retinal structure by using spectral-domain optical coherence tomography (SD-OCT) and retinal function using electroretinography (ERG) for nine months. We compared these to P23H RHO transgenic mice injected unilaterally with a control vector. Though retinas continued to thin over time, compared to control injected eyes, treatment with AAV-RHO820-shRNA820 slowed the loss of photoreceptor cells and the decrease in ERG amplitudes during the nine-month study period. Unexpectedly, we also observed the preservation of retinal structure and function in the untreated contralateral eyes of AAV-RHO820-shRNA820 treated mice. PCR analysis and western blots showed that a low amount of vector from injected eyes was present in uninjected eyes. In addition, protective neurotrophic factors bFGF and GDNF were elevated in both eyes of treated mice. Our finding suggests that using this or similar RNA replacement vectors in human gene therapy may provide clinical benefit to both eyes of patients with adRP.

Phytophthora sojae Transformation Based on the CRISPR/Cas9 System.

Phytophthora sojae is a model species for the study of plant pathogenic oomycetes. The initial research on gene function using Phytophthora was mainly based on gene silencing technology. Recently, the CRISPR/Cas9-mediated genome editing technology was successfully established in P. sojae and widely used in oomycetes. In this protocol, we describe the operating procedures for the use of CRISPR/Cas9-based genome editing technology and PEG-mediated stable transformation of P. sojae protoplasts. Two plasmids were co-transformed into P. sojae: pYF515 expressing Cas9 and the single guide RNA, and the homologous replacement vector of the candidate gene. Finally, the ORF of candidate gene were replaced with the ORF of the entire hygromycin B phosphotransferase gene (HPH), to achieve precise knockout.

Targeted Disruption of Scytalone Dehydratase Gene Using Agrobacterium tumefaciens-Mediated Transformation Leads to Altered Melanin Production in Ascochyta lentis.

Sustainable crop production is constantly challenged by the rapid evolution of fungal pathogens equipped with an array of host infection strategies and survival mechanisms. One of the devastating fungal pathogens that infect lentil is the ascomycete Ascochyta lentis which causes black spot or ascochyta blight (AB) on all above ground parts of the plant. In order to explore the mechanisms involved in the pathogenicity of A. lentis, we developed a targeted gene replacement method using Agrobacterium tumefaciens mediated transformation (ATMT) to study and characterize gene function. In this study, we investigated the role of scytalone dehydratase (SCD) in the synthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in AlKewell. Two SCD genes have been identified in AlKewell, AlSCD1 and AlSCD2. Phylogenetic analysis revealed that AlSCD1 clustered with the previously characterized fungal SCDs; thus, AlSCD1 was disrupted using the targeted gene replacement vector, pTAR-hyg-SCD1. The vector was constructed in a single step process using Gibson Assembly, which facilitated an easy and seamless assembly of multiple inserts. The resulting AlKewell scd1::hyg transformants appeared light brown/brownish-pink in contrast to the dark brown pycnidia of the WT strain and ectopic transformant, indicating an altered DHN-melanin production. Disruption of AlSCD1 gene did not result in a change in the virulence profile of AlKewell towards susceptible and resistant lentil varieties. This is the first report of a targeted gene manipulation in A. lentis which serves as a foundation for the functional gene characterization to provide a better understanding of molecular mechanisms involved in pathogen diversity and host specificity.

Development of Tomato bushy stunt virus-based vectors for fusion and non-fusion expression of heterologous proteins in an alternative host Nicotiana excelsiana.

Plant virus-based expression systems are an alternative expression platform for the production of clinically and industrially useful recombinant proteins. Nonetheless, due to a lack of viral vector with the commercial potentials, it is urgent to design and develop new, versatile, and efficient plant virus vectors. The genome of Tomato bushy stunt virus (TBSV) offers an attractive alternative to being modified as a vector for producing heterologous proteins in plants. Here, we developed a set of novel fusion and non-fusion TBSV-CP replacement vectors, which provide more flexible and efficient tools for expressing proteins of interest in plants. An alternative tobacco plant, Nicotiana excelsiana, was used in this study as a host for newly constructed TBSV vectors because the unwanted necrotic effects were reported on the commonly used Nicotiana benthamiana host associated with expression of TBSV-encoded P19 protein. The data showed that TBSV vectors caused a symptomless infection and overexpressed reporter gene in N. excelsiana leaves, demonstrating that N. excelsiana is an ideal host plant for TBSV-mediated heterologous gene expression. Moreover, a TBSV non-fusion vector, dAUG, shows the similar accumulation level of reporter proteins to that of TMV- and PVX-based vectors in side-by-side comparison and provides more flexible aspects than the previously developed TBSV vectors. Collectively, our newly developed TBSV expression system adds a new member to the family of plant viral expression vectors and meanwhile offers a flexible and highly effective approach for producing proteins of interest in plants. KEY POINTS: • The TBSV-based transient expression system has been significantly improved. • The necrotic effects caused by viral P19 protein were avoided by the usage of N. excelsiana as a host plant. • The expression level of the non-fusion vector was similar to the most effective virus vectors reported so far.

The Atypical Guanylate Kinase MoGuk2 Plays Important Roles in Asexual/Sexual Development, Conidial Septation, and Pathogenicity in the Rice Blast Fungus.

Guanylate kinases (GKs), which convert guanosine monophosphate into guanosine diphosphate (GDP), are important for growth and mannose outer chain elongation of cell wall N-linked glycoproteins in yeast. Here, we identified the ortholog of Saccharomyces cerevisiae GK Guk1, named MoGuk1 and a novel family of fungal GKs MoGuk2 in the rice blast fungus Magnaporthe oryzae. MoGuk1 contains 242 aa with an C-terminal GuKc domain that very similar to yeast Guk1. MoGuk2 contains 810 amino acids with a C-terminal GuKc domain and an additional N-terminal efThoc1 domain. Expression of either MoGuk1 or MoGuk2 in heterozygote yeast guk1 mutant could increase its GDP level. To investigate the biological role of MoGuk1 and MoGuk2 in M. oryzae, the gene replacement vectors were constructed. We obtained the ΔMoguk2 but not ΔMoguk1 mutant by screening over 1,000 transformants, indicating MoGuk1 might be essential for M. oryzae. The ΔMoguk2 mutant showed weak reductions in vegetative growth, conidial germination, appressorial formation, and appressorial turgor, and showed significant reductions in sporulation and pathogenicity. Moreover, the ΔMoguk2 mutant failed to produce perithecia and was sensitive to neomycin and a mixture of neomycin-tunicamycin. Exogenous GDP and ATP partially rescued the defects in conidial germination, appressorial formation, and infectious growth of the mutant. Further analysis revealed that intracellular GDP and GTP level was decreased, and GMP level was increased in the mutant, suggesting that MoGuk2 exhibits enzymatic activity. Structural analysis proved that the efThoc1, GuKc, and P-loop domains are essential for the full function of MoGuk2. Taken together, our data suggest that the guanylate kinase MoGuk2 is involved in the de novo GTP biosynthesis pathway and is important for infection-related morphogenesis in the rice blast fungus.

The Nep1-like protein family of Magnaporthe oryzae is dispensable for the infection of rice plants

The necrosis- and ethylene-inducing protein 1 (Nep1)-like proteins (NLPs) are a class of microbe-associated molecular patterns widely distributed across diverse groups of plant-associated microorganisms. In spite of the cytotoxic activity in dicot plants, the role of most NLPs in the virulence of plant pathogens is still largely unknown. We showed that the MoNLP family of rice blast fungus varied very little in amino acid sequence, transient expression of three MoNLPs induced cell death and the production of reactive oxygen species in Nicotiana benthamiana, and the expression of MoNLPs was induced during infection of susceptible rice plants. To further investigate the biological role of the MoNLP family, a marker-free gene replacement vector was developed and used to knock out the whole family in Magnaporthe oryzae. Results showed no significant difference in disease levels caused by wild type and the quadruple ΔMoNLP mutant strains. Likewise, the sporulation and radial growth of the two strains were similar under various unfavorable cultural conditions including malnutrition and abiotic stresses. These observations demonstrated that the MoNLP family is dispensable for the fungal tolerance to the tested adverse cultural conditions, and more importantly, for the virulence of blast fungus on susceptible rice plants.

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2.

RNAi-mediated TCR Knockdown Prevents Autoimmunity in Mice Caused by Mixed TCR Dimers Following TCR Gene Transfer

Genetically modified T cells that express a transduced T cell receptor (TCR) α/β heterodimer in addition to their endogenous TCR are used in clinical studies to treat cancer. These cells express two TCR-α and two TCR-β chains that do not only compete for CD3 proteins but also form potentially self-reactive mixed TCR dimers, composed of endogenous and transferred chains. To overcome these deficits, we developed an RNAi-TCR replacement vector that simultaneously silences the endogenous TCR and expresses an RNAi-resistant TCR. Transduction of the virus-specific P14 TCR without RNAi resulted in unequal P14 TCR-α and -β chain surface levels, indicating heterodimerization with endogenous TCR chains. Such unequal expression was also observed following TCR gene optimization. Equal surface levels of the introduced TCR chains were however achieved by silencing the endogenous TCR. Importantly, all mice that received cells transduced with the native or optimized P14 TCR developed lethal TCR gene transfer-induced graft-versus-host-disease (TI-GVHD) due to formation of mixed TCR dimers. In contrast, TI-GVHD was almost completely prevented when using the RNAi-TCR replacement vector. Our data demonstrate that RNAi-assisted TCR replacement reduces the formation of mixed TCR dimers, and thereby significantly reduces the risk of TI-GVHD in TCR gene therapy.

Tombusvirus-based vector systems to permit over-expression of genes or that serve as sensors of antiviral RNA silencing in plants

A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing.

Lower Citrinin Production by Gene Disruption of ctnB Involved in Citrinin Biosynthesis in Monascus aurantiacus Li AS3.4384

The filamentous fungi Monascus spp. have been used in the production of food colorants and health remedies for more than 1000 years in Asia. However, greater attention has been given to the safety of Monascus products because they contain citrinin, which is harmful to the hepatic and renal systems. The citrinin biosynthetic gene cluster has been characterized in Monasucs aurantiacus . The ctnB gene encoding an oxidoreductase is located between pksCT and ctnA. In this study, a ctnB replacement vector (pCTNB-HPH) was constructed to disrupt the ctnB gene with a hygromycin resistance gene as the selection marker. The linear vector was transformed into M. aurantiacus using the protoplast CaCl2/polyethylene glycol (PEG) method. Three ctnB-disrupted strains were obtained by homologous recombination. In comparison to the parental strain, the ΔctnB mutants barely produced citrinin. These data confirmed that the ctnB gene is directly involved in citrinin biosynthesis. Moreover, the yields of the pigments of two disruptants were similar to that of the wild-type strain, but the yield of another mutant was slightly higher than that of the latter strain. These results indicate that the production of the mycotoxin citrinin was successfully eliminated through genetic engineering.

Arrested Oocyst Maturation in Plasmodium Parasites Lacking Type II NADH:Ubiquinone Dehydrogenase

The Plasmodium mitochondrial electron transport chain has received considerable attention as a potential target for new antimalarial drugs. Atovaquone, a potent inhibitor of Plasmodium cytochrome bc(1), in combination with proguanil is recommended for chemoprophylaxis and treatment of malaria. The type II NADH:ubiquinone oxidoreductase (NDH2) is considered an attractive drug target, as its inhibition is thought to lead to the arrest of the mitochondrial electron transport chain and, as a consequence, pyrimidine biosynthesis, an essential pathway for the parasite. Using the rodent malaria parasite Plasmodium berghei as an in vivo infection model, we studied the role of NDH2 during Plasmodium life cycle progression. NDH2 can be deleted by targeted gene disruption and, thus, is dispensable for the pathogenic asexual blood stages, disproving the candidacy for an anti-malarial drug target. After transmission to the insect vector, NDH2-deficient ookinetes display an intact mitochondrial membrane potential. However, ndh2(-) parasites fail to develop into mature oocysts in the mosquito midgut. We propose that Plasmodium blood stage parasites rely on glycolysis as the main ATP generating process, whereas in the invertebrate vector, a glucose-deprived environment, the malaria parasite is dependent on an intact mitochondrial respiratory chain.

Development of expression vectors based on pepino mosaic virus.

BackgroundPlant viruses are useful expression vectors because they can mount systemic infections allowing large amounts of recombinant protein to be produced rapidly in differentiated plant tissues. Pepino mosaic virus (PepMV) (genus Potexvirus, family Flexiviridae), a widespread plant virus, is a promising candidate expression vector for plants because of its high level of accumulation in its hosts and the absence of severe infection symptoms. We report here the construction of a stable and efficient expression vector for plants based on PepMV.ResultsAgroinfectious clones were produced from two different PepMV genotypes (European and Chilean), and these were able to initiate typical PepMV infections. We explored several strategies for vector development including coat protein (CP) replacement, duplication of the CP subgenomic promoter (SGP) and the creation of a fusion protein using the foot-and-mouth disease virus (FMDV) 2A catalytic peptide. We found that CP replacement vectors were unable to move systemically and that vectors with duplicated SGPs (even heterologous SGPs) suffered from significant transgene instability. The fusion protein incorporating the FMDV 2A catalytic peptide gave by far the best results, maintaining stability through serial passages and allowing the accumulation of GFP to 0.2-0.4 g per kg of leaf tissue. The possible use of PepMV as a virus-induced gene silencing vector to study gene function was also demonstrated. Protocols for the use of this vector are described.ConclusionsA stable PepMV vector was generated by expressing the transgene as a CP fusion using the sequence encoding the foot-and-mouth disease virus (FMDV) 2A catalytic peptide to separate them. We have generated a novel tool for the expression of recombinant proteins in plants and for the functional analysis of virus and plant genes. Our experiments have also highlighted virus requirements for replication in single cells as well as intercellular and long-distance movement.

Suppression and Replacement Gene Therapy for Autosomal Dominant Disease in a Murine Model of Dominant Retinitis Pigmentosa

For dominantly inherited disorders development of gene therapies, targeting the primary genetic lesion has been impeded by mutational heterogeneity. An example is rhodopsin-linked autosomal dominant retinitis pigmentosa with over 150 mutations in the rhodopsin gene. Validation of a mutation-independent suppression and replacement gene therapy for this disorder has been undertaken. The therapy provides a means of correcting the genetic defect in a mutation-independent manner thereby circumventing the mutational diversity. Separate adeno-associated virus (AAV) vectors were used to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site. Viruses were subretinally coinjected into P347S mice, a model of dominant rhodopsin-linked retinitis pigmentosa. Benefit in retinal function and structure detected by electroretinography (ERG) and histology, respectively, was observed for at least 5 months. Notably, the photoreceptor cell layer, absent in 5-month-old untreated retinas, contained 3–4 layers of nuclei, whereas photoreceptor ultrastructure, assessed by transmission electron microscopy (TEM) improved significantly. The study provides compelling evidence that codelivered suppression and replacement is beneficial, representing a significant step toward the clinic. Additionally, dual-vector delivery of combined therapeutics represents an exciting approach, which is potentially applicable to other inherited disorders.

PS3-23 In human multiple sclerosis, T helper 17 lymphocytes display increased levels of JAK2 that stabilize cell surface IFN-γR2 chain

Genetically modified T cells that express a transduced T cell receptor (TCR) α/β heterodimer in addition to their endogenous TCR are used in clinical studies to treat cancer. These cells express two TCR-α and two TCR-β chains that do not only compete for CD3 proteins but also form potentially self-reactive mixed TCR dimers, composed of endogenous and transferred chains. To overcome these deficits, we developed an RNAi-TCR replacement vector that simultaneously silences the endogenous TCR and expresses an RNAi-resistant TCR. Transduction of the virus-specific P14 TCR without RNAi resulted in unequal P14 TCR-α and -β chain surface levels, indicating heterodimerization with endogenous TCR chains. Such unequal expression was also observed following TCR gene optimization. Equal surface levels of the introduced TCR chains were however achieved by silencing the endogenous TCR. Importantly, all mice that received cells transduced with the native or optimized P14 TCR developed lethal TCR gene transfer-induced graft-versus-host-disease (TI-GVHD) due to formation of mixed TCR dimers. In contrast, TI-GVHD was almost completely prevented when using the RNAi-TCR replacement vector. Our data demonstrate that RNAi-assisted TCR replacement reduces the formation of mixed TCR dimers, and thereby significantly reduces the risk of TI-GVHD in TCR gene therapy.

Inflammatory and age-related pathologies in mice with ectopic expression of human PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) is a sensor for DNA strand breaks and some unusual DNA structures and catalyzes poly(ADP-ribosyl)ation of nuclear proteins with NAD + serving as substrate. PARP-1 is involved in the regulation of genomic integrity, transcription, inflammation, and cell death. Due to its versatile role, PARP-1 is discussed both as a longevity factor and as an aging-promoting factor. Recently, we generated a mouse model with ectopic integration of full-length hPARP-1 [Mangerich, A., Scherthan, H., Diefenbach, J., Kloz, U., van der Hoeven, F., Beneke, S. and Bürkle, A., 2009. A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1. Transgenic Res. 18, 261–279]. Here, we show that hPARP-1 mice exhibit impaired survival rates accompanied by reduced hair growth and premature development of several inflammation and age-associated pathologies, such as adiposity, kyphosis, nephropathy, dermatitis, pneumonitis, cardiomyopathy, hepatitis, and anemia. Moreover, mutant male mice showed impaired glucose tolerance, yet without developing manifest diabetes. Overall tumor burden was comparable in wild-type and hPARP-1 mice, but tumor spectrum was shifted in mutant mice, showing lower incidence of sarcomas, but increased incidence of carcinomas. Furthermore, DNA repair was delayed in splenocytes of hPARP-1 mice, and gene expression of pro-inflammatory cytokines was dysregulated. Our results suggest that in hPARP-1 mice impaired DNA repair, accompanied by a continuous low-level increase in pro-inflammatory stimuli, causes development of chronic diseases leading to impaired survival.

Impact of the RNA chaperone Hfq on multidrug resistance in Escherichia coli

Hfq is a bacterial RNA chaperone involved in the post-transcriptional regulation of many stress-inducible genes via small non-coding RNAs. Although Hfq is related to important phenotypes including virulence in many bacterial pathogens, its role in drug resistance is unknown. The aim of this study was to investigate the role of Hfq in bacterial multidrug resistance. The hfq gene was inactivated in Escherichia coli by use of pKO3, which is a gene replacement vector. The drug susceptibility and drug accumulation of the hfq mutant were determined. The level of production of the AcrB multidrug efflux pump in this mutant was also measured. The hfq mutant was susceptible to acriflavine, benzalkonium, cefamandole, chloramphenicol, Crystal Violet, nalidixic acid, novobiocin, oxacillin and rhodamine 6G. E. coli cells were strongly stained with rhodamine 6G compared with the wild-type on deletion of hfq, indicating that Hfq affects the accumulation of the drug in bacterial cells. The deletion of the drug efflux gene acrB impairs the effect of hfq deletion on E. coli susceptibility. Furthermore, the level of AcrB protein production was reduced in the hfq mutant, whereas hfq deletion did not affect the promoter activity of the acrAB operon. These results indicate that Hfq regulates the drug efflux system at the post-transcriptional level and reveals the previously uncharacterized role of Hfq in bacterial multidrug resistance.

Adeno-Associated Virus-Mediated Rhodopsin Replacement Provides Therapeutic Benefit in Mice with a Targeted Disruption of the Rhodopsin Gene

The rhodopsin gene (RHO) encodes a highly expressed G protein-coupled receptor that is central to visual transduction in rod photoreceptors. A suite of recombinant 2/5 adeno-associated viral (AAV) RHO replacement vectors has been generated in an attempt to recapitulate endogenous rhodopsin levels from exogenously delivered AAV vectors in the retina of mice with a targeted disruption in the rhodopsin gene (Rho(-/-) mice). Approximately 40% of wild-type mouse rhodopsin mRNA levels (RNA taken from whole retinas) was achieved in vivo in AAV-RHO-injected eyes, representing approximately 50-fold increases in expression compared with the initial vector. The main focus of this study was to test whether expression of AAV-RHO replacement in Rho(-/-) mice provided therapeutic benefit, which to date had not been achieved. Rho(-/-) mice neither elaborate rod outer segments nor have rod-derived electroretinograms (ERGs). Our results indicate for the first time in this model that subretinal AAV-RHO delivery leads not only to RHO immunolabeling but the generation of rod outer segments as evaluated by light and transmission electron microscopy. Improved histology was accompanied by rod photoreceptor activity as assessed by ERG for at least 12 weeks postinjection. The most efficient AAV-RHO constructs presented in this study provide sufficient levels of RHO to be of therapeutic benefit in Rho(-/-) mice and therefore represent important steps toward generating potent AAV-RHO replacement genes for gene therapy in RHO-linked human retinopathies.

Critical Roles of Subunit NuoH (ND1) in the Assembly of Peripheral Subunits with the Membrane Domain of Escherichia coli NDH-1

The bacterial proton-translocating NADH:quinone oxidoreductase (NDH-1) consists of two domains, a peripheral arm and a membrane arm. NuoH is a counterpart of ND1, which is one of seven mitochondrially encoded hydrophobic subunits, and is considered to be involved in quinone/inhibitor binding. Sequence comparison in a wide range of species showed that NuoH is comprehensively conserved, particularly with charged residues in the cytoplasmic side loops. We have constructed 40 mutants of 27 conserved residues predicted to be in the cytoplasmic side loops of Escherichia coli NuoH by utilizing the chromosomal DNA manipulation technique and investigated roles of these residues. Mutants of Arg(37), Arg(46), Asp(63), Gly(134), Gly(145), Arg(148), Glu(220), and Glu(228) showed low deamino-NADH-K(3)Fe(CN)(6) reductase activity, undetectable NDH-1 in Blue Native gels, low contents of peripheral subunits (especially NuoB and NuoCD) bound to the membranes, and a significant loss of the membrane potential and proton-pumping function coupled to deamino-NADH oxidation. The results indicated that these conserved residues located in the cytoplasmic side loops are essential for the assembly of the peripheral subunits with the membrane arm. Implications for the involvement of NuoH (ND1) in maintaining the structure and function of NDH-1 are discussed.