scholarly journals Chimeric Capsid Proteins Impact Transduction Efficiency of Haploid Adeno-Associated Virus Vectors

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1138
Author(s):  
Zheng Chai ◽  
Xintao Zhang ◽  
Amanda Lee Dobbins ◽  
Ellie Azure Frost ◽  
R. Jude Samulski ◽  
...  
Keyword(s):  
Fold Increase ◽  
Transduction Efficiency ◽  
Adeno Associated Virus ◽  
Systemic Injection ◽  
Copy Numbers ◽  
C Terminus ◽  
Vector Genome ◽  
Significant Difference ◽  
N Terminus ◽  
The Difference

Our previous studies have demonstrated that haploid AAV vectors made from capsids of two different serotypes induced high transduction and prevented serotype-specific antibody binding. In this study, we explored the transduction efficiency of several haploid viruses, which were made from the VP1/VP2 of one serotype and VP3 of another compatible serotype. After systemic injection of 2 × 1010 vg of AAV vectors into mice, the haploid AAV vectors, composed of VP1/VP2 from serotypes 8 or 9, and VP3 from AAV2, displayed a two to seven-fold increase in liver transduction compared with those of parental AAV2 vectors. Furthermore, a chimeric AAV2/8 VP1/VP2 with N-terminus of VP1/VP2 from AAV2 and C-terminus (VP3 domain) from AAV8 was constructed, and produced the haploid vector 28m-2VP3 with AAV2 VP3. The haploid 28m-2VP3 vector showed a five-fold higher transduction than that of the vectors composed solely of AAV2 VPs. Remarkably, the 28m-2VP3 vectors also induced a significant increase in transgene expression compared to the vectors composed of AAV8 VP1/VP2 with AAV2 VP3. The results suggest that the difference in the VP1/VP2 N-terminal region between AAV2 and AAV8 may allow better “communication” between the VP1/VP2 N-terminus of AAV2 with its cognate VP3. Similarly, the haploid vectors, VP1/VP2 from serotypes 8 or 9 and VP3 from AAV3, achieved higher transductions in multiple tissue types beyond typical tropism compared with those of AAV3 vectors. Consistently, higher vector genome copy numbers were detected in these tissues, indicating that an incorporation of non-cognate VP1/VP2 might influence the cellular tropism of the haploid vectors. However, there was no significant difference or even decreased transductions when compared with those of parental AAV8 or AAV9 vectors. In summary, these studies provide insight into current development strategies of AAV vectors that can increase AAV transduction across multiple tissues.

scholarly journals An Exon-Specific Small Nuclear U1 RNA (ExSpeU1) Improves Hepatic OTC Expression in a Splicing-Defective spf/ash Mouse Model of Ornithine Transcarbamylase Deficiency

2020 ◽  
Vol 21 (22) ◽  
pp. 8735
Author(s):  
Dario Balestra ◽  
Mattia Ferrarese ◽  
Silvia Lombardi ◽  
Nicole Ziliotto ◽  
Alessio Branchini ◽  
...  
Keyword(s):  
Mouse Model ◽  
Protein Function ◽  
Disease Onset ◽  
Fold Increase ◽  
Ornithine Transcarbamylase ◽  
Transduction Efficiency ◽  
Ornithine Transcarbamylase Deficiency ◽  
Adeno Associated Virus ◽  
Early Proof

OTC splicing mutations are generally associated with the severest and early disease onset of ornithine transcarbamylase deficiency (OTCD), the most common urea cycle disorder. Noticeably, splicing defects can be rescued by spliceosomal U1snRNA variants, which showed their efficacy in cellular and animal models. Here, we challenged an U1snRNA variant in the OTCD mouse model (spf/ash) carrying the mutation c.386G > A (p.R129H), also reported in OTCD patients. It is known that the R129H change does not impair protein function but affects pre-mRNA splicing since it is located within the 5′ splice site. Through in vitro studies, we identified an Exon Specific U1snRNA (ExSpeU1O3) that targets an intronic region downstream of the defective exon 4 and rescues exon inclusion. The adeno-associated virus (AAV8)-mediated delivery of the ExSpeU1O3 to mouse hepatocytes, although in the presence of a modest transduction efficiency, led to increased levels of correct OTC transcripts (from 6.1 ± 1.4% to 17.2 ± 4.5%, p = 0.0033). Consistently, this resulted in increased liver expression of OTC protein, as demonstrated by Western blotting (~3 fold increase) and immunostaining. Altogether data provide the early proof-of-principle of the efficacy of ExSpeU1 in the spf/ash mouse model and encourage further studies to assess the potential of RNA therapeutics for OTCD caused by aberrant splicing.

scholarly journals Separate bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: the role of the C-terminal tail in modulating enzyme activity

1997 ◽  
Vol 328 (3) ◽  
pp. 751-756 ◽  
Author(s):  
Lin LI ◽  
Song LING ◽  
Chun-lai WU ◽  
Wei-zhe YAO ◽  
Gen-jun XU
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Substrate Inhibition ◽  
Fold Increase ◽  
Chicken Liver ◽  
Bifunctional Enzyme ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Km Value ◽  
The Difference

The separate bisphosphatase domain (amino acid residues 243-468) of the chicken liver bifunctional enzyme 6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase was expressed in Escherichia coli and purified to homogeneity. The fructose-2,6-bisphosphatase activity of the separate domain was 7-fold higher than that of the native bifunctional enzyme, and exhibited substrate inhibition characteristic of the native enzyme. The inhibition of the enzymes by fructose 2,6-bisphosphate could be overcome by Pi, glycerol 3-phosphate and GTP. Deletion of 30 amino acid residues from the C-terminus of the separate domain resulted in around a 5-fold increase in the Vmax of the bisphosphatase. Also, the truncated form was more accessible to chemical modification by diethyl pyrocarbonate and N-ethylmaleimide, suggesting a more open structure than the wild-type form. In addition, the mutation of cysteine-389 to alanine increased bisphosphatase activity by 20% and the Km value for fructose 2,6-bisphosphate by 3-fold, and both the point mutation at cysteine-389 and the deletional mutation led to the predominantly insoluble expression of the enzyme. The results indicated that the C-terminal tail plays a role in modulating the enzyme activity and suggested that the difference in the C-terminal tail sequence is responsible for the difference in activity of the chicken and rat liver fructose-2,6-bisphosphatases. It is postulated that an interaction between the C-terminal tail and the active site might be present.

scholarly journals Cotranslational Protein Folding and Terminus Hydrophobicity

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Sheenal Srivastava ◽  
Yumi Patton ◽  
David W. Fisher ◽  
Graham R. Wood
Keyword(s):  
Structure Prediction ◽  
Square Lattice ◽  
Prediction Program ◽  
C Terminus ◽  
Cotranslational Folding ◽  
N Terminus ◽  
Structure Prediction Program ◽  
The Difference ◽  
Logarithmic Ratio ◽  
Time Required

Peptides fold on a time scale that is much smaller than the time required for synthesis, whence all proteins potentially fold cotranslationally to some degree (followed by additional folding events after release from the ribosome). In this paper, in three different ways, we find that cotranslational folding success is associated with higher hydrophobicity at the N-terminus than at the C-terminus. First, we fold simple HP models on a square lattice and observe that HP sequences that fold better cotranslationally than from a fully extended state exhibit a positive difference (N−C) in terminus hydrophobicity. Second, we examine real proteins using a previously established measure of potential cotranslationality known as ALR (Average Logarithmic Ratio of the extent of previous contacts) and again find a correlation with the difference in terminus hydrophobicity. Finally, we use the cotranslational protein structure prediction program SAINT and again find that such an approach to folding is more successful for proteins with higher N-terminus than C-terminus hydrophobicity. All results indicate that cotranslational folding is promoted in part by a hydrophobic start and a less hydrophobic finish to the sequence.

scholarly journals Involvement of Cellular Double-Stranded DNA Break Binding Proteins in Processing of the Recombinant Adeno-Associated Virus Genome

2001 ◽  
Vol 75 (24) ◽  
pp. 12279-12287 ◽  
Author(s):  
Lorena Zentilin ◽  
Alessandro Marcello ◽  
Mauro Giacca
Keyword(s):  
Homologous Recombination ◽  
Cultured Cells ◽  
Virus Genome ◽  
Nonhomologous End Joining ◽  
Transduction Efficiency ◽  
End Joining ◽  
Adeno Associated Virus ◽  
Vector Genome ◽  
Recombination Pathway

ABSTRACT Unlike postmitotic tissues in vivo, transduction of cultured cells is poor with recombinant adeno-associated virus (rAAV). The ability of rAAV to transduce cells is greatly enhanced by a variety of agents that induce DNA damage and is elevated in cells defective in the ataxia telangiectasia gene product (ATM), showing increased genomic instability. Here we show that DNA double-stranded break (DSB) repair pathways are involved in the regulation of rAAV transduction efficiency. By quantitative chromatin immunoprecipitation, we found that Ku86 and Rad52 proteins associate with viral DNA inside transduced cells. Both proteins are known to competitively recognize hairpin structures and DNA termini and to promote repair of DSBs, the former by facilitating nonhomologous end joining and the latter by initiating homologous recombination. We found that rAAV transduction is increased in Ku86-defective cells while it is inhibited in Rad52 knockout cells. These results suggest that binding of Rad52 to the rAAV genome might be involved in processing of the vector genome through a homologous recombination pathway.

scholarly journals Transient cytochalasin-D treatment induces apically administered rAAV2 across tight junctions for transduction of enterocytes

2008 ◽  
Vol 89 (12) ◽  
pp. 3004-3008 ◽  
Author(s):  
Ya-Yuan Fu ◽  
Eric Sibley ◽  
Shiue-Cheng Tang
Keyword(s):  
Tight Junctions ◽  
Actin Filaments ◽  
Transgene Expression ◽  
Fold Increase ◽  
Cytochalasin D ◽  
Transduction Efficiency ◽  
Intestinal Epithelial ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Virus Serotype

Enteropathogens are known to disrupt apical actin filaments and/or tight-junction barriers of intestinal epithelial cells to promote infection. In this study, we show that a controlled, cytochalasin-D (Cyto-D)-mediated disruption of actin filaments and tight junctions enhanced the apical delivery of the gene-therapy vector recombinant adeno-associated virus serotype 2 (rAAV2). This increase in transduction efficiency can be attributed to the enhanced delivery of rAAV2 across the Cyto-D disrupted tight junctions, allowing basolateral entry of rAAV2. Previously, we have shown that MG101 and doxorubicin are capable of overcoming proteasome-mediated transduction barriers of rAAV2 in enterocytes. In this study, when Cyto-D was combined with MG101 and doxorubicin in apical delivery of rAAV2 to transduce the differentiated Caco-2 enterocytes, a synergistic >2300-fold increase in transgene expression was achieved. We conclude that Cyto-D is capable of permeating the polarized enterocytes for rAAV2 transduction, which may potentially be a useful device to facilitate intestinal gene transfer via the gut lumen.

scholarly journals Plasmid DNA Sequences Present in Conventional Herpes Simplex Virus Amplicon Vectors Cause Rapid Transgene Silencing by Forming Inactive Chromatin

2006 ◽  
Vol 80 (7) ◽  
pp. 3293-3300 ◽  
Author(s):  
Masataka Suzuki ◽  
Kazue Kasai ◽  
Yoshinaga Saeki
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Sequences ◽  
Transgene Expression ◽  
Human Fibroblasts ◽  
Transcriptional Level ◽  
Copy Numbers ◽  
Vector Genome ◽  
Significant Difference ◽  
Simplex Virus

ABSTRACT The herpes simplex virus (HSV)-based amplicon vector, a bacterial-viral-mammalian cell shuttle system, holds promise as a versatile gene delivery vehicle because of its large transgene capacity. However, amplicon-mediated transgene expression is often transient. We hypothesized that the presence of prokaryotic DNA sequences within the packaged vector genome can trigger transcriptional silencing of the entire vector sequence. To test this, we constructed a novel amplicon vector devoid of bacterial sequences (minicircle [MC] amplicon). Although the same dose of the minicircle amplicon vector in normal human fibroblasts resulted in an expression of luciferase approximately 20 times higher than that caused by the conventional amplicon vector, no significant difference was observed in copy numbers of luciferase DNA between MC amplicon- and control-transduced cells. Quantitative analyses of levels of luciferase mRNA revealed that differential expression of luciferase was controlled at the transcriptional level. Chromatin immunoprecipitation PCR analyses of several regions of vector genomes revealed that the bacterial sequences found in the conventional amplicon DNA were associated with an inactive form of chromatin immediately after infection. The presence of bacterial sequences also affected the remaining vector sequences in the conventional amplicon vector. Finally, nude mice injected with the MC amplicon exhibited higher and more sustained expression of luciferase than those injected with the conventional amplicon, confirming the usefulness of the MC amplicon devoid of bacterial sequences. Although additional improvements are absolutely required, these findings are a significant first step toward developing a novel HSV amplicon vector that can achieve enhanced long-term transgene expression.

Pharmacokinetics of Full Length and Two-Domain Tissue Factor Pathway Inhibitor in Combination with Heparin in Rabbits

1993 ◽  
Vol 70 (03) ◽  
pp. 454-457 ◽  
Author(s):  
Claus Bregengaard ◽  
Ole Nordfang ◽  
Per Østergaard ◽  
Jens G L Petersen ◽  
Giorgio Meyn ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Anticoagulant Activity ◽  
Fold Increase ◽  
Volume Of Distribution ◽  
Full Length ◽  
Heparin Binding ◽  
Extrinsic Pathway ◽  
C Terminus ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a feed back inhibitor of the initial activation of the extrinsic pathway of coagulation. In humans, injection of heparin results in a 2-6 fold increase in plasma TFPI and recent studies suggest that TFPI may be important for the anticoagulant activity of heparin. Full length (FL) TFPI, but not recombinant two-domain (2D) TFPI, has a poly cationic C-terminus showing very strong heparin binding. Therefore, we have investigated if heparin affects the pharmacokinetics of TFPI with and without this C-terminus.FL-TFPI (608 U/kg) and 2D-TFPI (337 U/kg) were injected intravenously in rabbits with and without simultaneous intravenous injections of low molecular weight heparin (450 anti-XaU/kg).Heparin decreased the volume of distribution and the clearance of FL-TFPI by a factor 10-15, whereas the pharmacokinetics of 2D-TFPI were unaffected by heparin. When heparin was administered 2 h following TFPI the recovery of FL-TFPI was similar to that found in the group receiving the two compounds simultaneously, suggesting that the releasable pool of FL-TFPI is removed very slowly in the absence of circulating heparin.

Cranial crassicaudiasis in two coastal dolphin species from South Africa is predominantly a disease of immature individuals

2020 ◽  
Vol 139 ◽  
pp. 93-102 ◽  
Author(s):  
MF Van Bressem ◽  
P Duignan ◽  
JA Raga ◽  
K Van Waerebeek ◽  
N Fraijia-Fernández ◽  
...  
Keyword(s):  
South Africa ◽  
Bone Development ◽  
Bottlenose Dolphins ◽  
Fatal Outcome ◽  
Population Level ◽  
Cape Coast ◽  
Osteolytic Lesions ◽  
Immune Mediated ◽  
Significant Difference ◽  
The Difference

Crassicauda spp. (Nematoda) infest the cranial sinuses of several odontocetes, causing diagnostic trabecular osteolytic lesions. We examined skulls of 77 Indian Ocean humpback dolphins Sousa plumbea and 69 Indo-Pacific bottlenose dolphins Tursiops aduncus, caught in bather-protecting nets off KwaZulu-Natal (KZN) from 1970-2017, and skulls of 6 S. plumbea stranded along the southern Cape coast in South Africa from 1963-2002. Prevalence of cranial crassicaudiasis was evaluated according to sex and cranial maturity. Overall, prevalence in S. plumbea and T. aduncus taken off KZN was 13 and 31.9%, respectively. Parasitosis variably affected 1 or more cranial bones (frontal, pterygoid, maxillary and sphenoid). No significant difference was found by gender for either species, allowing sexes to be pooled. However, there was a significant difference in lesion prevalence by age, with immature T. aduncus 4.6 times more likely affected than adults, while for S. plumbea, the difference was 6.5-fold. As severe osteolytic lesions are unlikely to heal without trace, we propose that infection is more likely to have a fatal outcome for immature dolphins, possibly because of incomplete bone development, lower immune competence in clearing parasites or an over-exuberant inflammatory response in concert with parasitic enzymatic erosion. Cranial osteolysis was not observed in mature males (18 S. plumbea, 21 T. aduncus), suggesting potential cohort-linked immune-mediated resistance to infestation. Crassicauda spp. may play a role in the natural mortality of S. plumbea and T. aduncus, but the pathogenesis and population level impact remain unknown.

Perbedaan Penggunaan Antikoagulan K2EDTA DAN K3EDTA Terhadap Hasil Pemeriksaan Indeks Eritrosit

2018 ◽  
Vol 1 (2) ◽  
pp. 114
Author(s):  
Wahdaniah Wahdaniah ◽  
Sri Tumpuk
Keyword(s):  
Screening Test ◽  
Venous Blood ◽  
The Body ◽  
Ethylene Diamine ◽  
P Value ◽  
Cross Sectional ◽  
Significant Difference ◽  
Ethylene Diamine Tetra Acetate ◽  
The Difference ◽  
Index Value

Abstract: Routine blood examination is the earliest blood test or screening test to determine the diagnosis of an abnormality. Blood easily froze if it is outside the body and can be prevented by the addition of anticoagulants, one of which Ethylene Diamine Tetra Acetate (EDTA). Currently available vacuum tubes containing EDTA anticoagulants in the form of K2EDTA and K3EDTA. K3EDTA is usually a salt that has better stability than other EDTA salts because it shows a pH approaching a blood pH of about 6.4. The purpose of this research is to know the difference of erythrocyte index results include MCH, MCV and MCHC using K3EDTA anticoagulant with K2EDTA. This research is a cross sectional design. This study used venous blood samples mixed with K2EDTA anticoagulant and venous blood mixed with K3EDTA anticoagulants, each of 30 samples. Data were collected and analyzed using paired different test. Based on data analysis that has been done on MCH examination, p value <0,05 then there is a significant difference between samples with K3EDTA anticoagulant with K2EDTA to erythrocyte index value. Then on the examination of MCV and MCHC obtained p value <0.05 then there is no significant difference between samples with K3EDTA anticoagulant with K2EDTA to erythrocyte index value.Abstrak: Pemeriksaan darah rutin merupakan pemeriksaan darah yang paling awal atau screening test untuk mengetahui diagnosis suatu kelainan. Darah mudah membeku jika berada diluar tubuh dan bisa dicegah dengan penambahan antikoagulan, salah satunya Ethylene Diamine Tetra Acetate (EDTA). Dewasa ini telah tersedia tabung vakum yang sudah berisi antikoagulan EDTA dalam bentuk  K2EDTA dan  K3EDTA. K3EDTA  biasanya berupa garam yang mempunyai stabilitas yang lebih baik dari garam EDTA yang lain karena menunjukkan pH yang mendekati pH darah yaitu sekitar 6,4. Tujuan dari penelitian ini adalah untuk mengetahui perbedaan hasil indeks eritrosit meliputi MCH, MCV dan MCHC menggunakan antikoagulan K3EDTA dengan K2EDTA. Penelitian ini merupakan penelitian dengan desain cross sectional. Penelitian ini menggunakan sampel darah vena yang dicampur dengan antikoagulan K2EDTA dan darah vena yang dicampur dengan antikoagulan K3EDTA, masing-masing sebanyak 30 sampel. Data dikumpulkan dan dianalisis menggunakan uji beda berpasangan. Berdasarkan analisis data yang telah dilakukan pada pemeriksaan MCH didapatkan nilai p < 0,05 maka ada perbedaan yang signifikan antara sampel dengan antikoagulan K3EDTA dengan K2EDTA terhadap nilai indeks eritrosit. Kemudian pada pemeriksaan MCV dan MCHC didapatkan nilai p < 0,05 maka tidak ada perbedaan yang signifikan antara sampel dengan antikoagulan K3EDTA dengan K2EDTA terhadap nilai indeks eritrosit.

Pengaruh Jumlah Pencucian Beras dengan Kadar Klorin

2017 ◽  
Vol 1 (1) ◽  
pp. 89
Author(s):  
Indah Purwaningsih ◽  
Supriyanto Supriyanto
Keyword(s):  
Mucous Membrane ◽  
Confidence Level ◽  
Iodometric Titration ◽  
Titration Method ◽  
Anova Test ◽  
Average Value ◽  
Significant Difference ◽  
The Difference

Abstract: Chlorine is a green halogen-shaped halogen gas at normal temperature and serves as bleach, stain remover and disinfectant. Chlorine is now widely used for bleaching rice so that less quality rice looks like quality rice. Chlorine is very toxic and causes mucous membrane irritation, highly reactive and very powerful oxidizer. The purpose of this research was to determine the difference of chlorine level in chlorinated rice washed once, twice and 3 times. The sample in this study amounted to 11 samples calculated by replication formula. Each sample was treated 3 times, ie 1 washed once, 2 washed twice and washed 3 times. The samples then examined by iodometric titration method. Based on the results of the study using ANOVA test, 11 samples obtained the average value of chlorine after washed once amount of 0.0176%, after washed twice amount of 0.0111%, and after washed 3 times amount of 0.0052% with the value significance p = 0.03 (p <0.05) at 95% confidence level which means there was a significant difference between chlorine levels in chlorinated rice washed once, twice and 3 times.Abstrak: Klorin merupakan unsur halogen berbentuk gas berwarna kuning kehijauan pada suhu normal danberfungsi sebagai pemutih, penghilang noda maupun desinfektan. Klorin sekarang banyak digunakan untuk bahan pemutih beras agar beras yang kurang berkualitas tampak seperti beras berkualitas. Klorin sangat toksik dan menyebabkan iritasi membran mukosa, sangat reaktif dan merupakan oksidator yang sangat kuat. Tujuan dari penelitian ini ialah untuk mengetahui perbedaan kadar klorin pada beras berklorin yang dicuci sebanyak 1 kali, 2 kali dan 3 kali. Sampel dalam penelitian ini berjumlah 11 sampel yang dihitung dengan rumus replikasi. Setiap sampel diberi perlakuan sebanyak 3 kali, yaitu 1 kali pencucian, 2 kali pencucian dan 3 kali pencucian. Sampel penelitian kemudian diperiksa dengan metode titrasi iodometri. Berdasarkan hasil penelitian menggunakan uji Anova secara komputerisasi terhadap 11 sampel diperoleh nilai rata-rata kadar klorin setelah 1 kali pencucian sebesar 0,0176 %, setelah 2 kali pencucian sebesar 0,0111 %, dan setelah 3 kali pencucian sebesar 0,0052 % dengan nilai signifkansi p = 0,03 (p<0,05) pada tingkat kepercayaan 95% yang artinya ada perbedaan yang bermakna antara kadar klorin pada beras berklorin yang dicuci sebanyak 1 kali, 2 kali dan 3 kali.

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