The effect of physical activity on mother-child relationship and parental attitudes: a follow-up study examining the long-term effects of COVID-19.

OBJECTIVE: This study was planned to evaluate effects of game-based physical activity model on mother-child relationship and parental attitudes during the prolonged COVID-19 pandemic period. SUBJECTS AND METHODS: This study was designed using a web-based quasi-experimental model with a pre-test/post-test evaluation, with a control group. The mothers who accepted to participate in the study and their children were divided into experimental (group I, n=28) and control groups (group II, n=31). The mothers and children in the experimental group were asked to apply web-based game-based physical activity model for 20 minutes/day for 4 weeks. The online questionnaire included socio-demographic data form, Child Parent Relationship Scale (CPRS), and Parental Attitude Scale (PAS). RESULTS: There were no significant differences between mean scores of pre-test and post-test subscales of the PAS in group I (p>0.05 for all subscales). It was found that post-test scores of democratic subscales of PAS statistically significant decreased (p=0.047) and the authoritarian attitude subscale scores significantly increased (p=0.033) in group II. The mean pre- and post-activity scores of positive/close relationship and conflictual relationship subscales of CPRS differ between groups (p<0.05 for both subscales). Pre-post test scores of group II were found to be significantly lower compared to group II. CONCLUSIONS: Our study provides a moderate improvement in parameters evaluated; however, we suggest that longer-term activities may have a more permanent and statistically significant effect.

Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.

Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into ∼0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.

Upregulation of the microRNA cluster at the Dlk1-Dio3 locus in lung adenocarcinoma.

Mice in which lung epithelial cells can be induced to express an oncogenic Kras(G12D) develop lung adenocarcinomas in a manner analogous to humans. A myriad of genetic changes accompany lung adenocarcinomas, many of which are poorly understood. To get a comprehensive understanding of both the transcriptional and post-transcriptional changes that accompany lung adenocarcinomas, we took an omics approach in profiling both the coding genes and the non-coding small RNAs in an induced mouse model of lung adenocarcinoma. RNAseq transcriptome analysis of Kras(G12D) tumors from F1 hybrid mice revealed features specific to tumor samples. This includes the repression of a network of GTPase-related genes (Prkg1, Gnao1 and Rgs9) in tumor samples and an enrichment of Apobec1-mediated cytosine to uridine RNA editing. Furthermore, analysis of known single-nucleotide polymorphisms revealed not only a change in expression of Cd22 but also that its expression became allele specific in tumors. The most salient finding, however, came from small RNA sequencing of the tumor samples, which revealed that a cluster of ∼53 microRNAs and mRNAs at the Dlk1-Dio3 locus on mouse chromosome 12qF1 was markedly and consistently increased in tumors. Activation of this locus occurred specifically in sorted tumor-originating cancer cells. Interestingly, the 12qF1 RNAs were repressed in cultured Kras(G12D) tumor cells but reactivated when transplanted in vivo. These microRNAs have been implicated in stem cell pleuripotency and proteins targeted by these microRNAs are involved in key pathways in cancer as well as embryogenesis. Taken together, our results strongly imply that these microRNAs represent key targets in unraveling the mechanism of lung oncogenesis.

Prevention of spontaneous bleeding in dogs with haemophilia A and haemophilia B.

Dogs with haemophilia A or haemophilia B exhibit spontaneous bleeding comparable with the spontaneous bleeding phenotype that occurs in humans with severe haemophilia. The phenotypic and genotypic characteristics of haemophilic dogs have been well-described, and such dogs are suitable for testing prophylactic protein replacement therapy and gene transfer strategies. In dogs with haemophilia, long-term effects on spontaneous bleeding frequency (measured over years) can be used as an efficacy endpoint in such studies. Although complete correction of coagulopathy has not been achieved, published data show that prophylactic factor replacement therapy and gene transfer can markedly reduce the frequency of spontaneous bleeding in haemophilic dogs. Further studies are currently ongoing.

Therapeutic short hairpin RNA expression in the liver: viral targets and vectors.

Over 500 million people worldwide are infected with one or more different and unrelated types of human hepatitis virus. Such individuals are at a high risk of developing acute or chronic hepatic disease, and ultimately dying from sequelae. Although a vaccine is available for hepatitis A and B virus, treatment options for chronically infected patients are limited, and particularly ineffective in case of hepatitis C virus (HCV) infection. A promising new avenue currently being explored is to harness the power of RNA interference for development of an antiviral therapy. The timing to pursue this particular approach is excellent, with the first in vivo animal models for HCV infection becoming available, and the technology for liver-specific expression of short hairpin RNAs advancing at a rapid pace. Here, we critically review these important current developments, and discuss the next steps to bring this novel approach into the clinics.

Genomic progression in mouse models for liver tumors.

The principal cause of human liver cancer is infection with hepatitis viruses B and C, but tumor progression is fueled by ensuing perturbations that confer gain of function on proto-oncogenes or loss of function on tumor suppressor genes. Frequent among these perturbations is overexpression of the proto-oncogene MET. We have modeled the pathogenesis of liver tumors by expressing conditional transgenes of MET in the hepatocytes of inbred mice. The response to the MET transgene varied with both the magnitude and timing of its expression but included hyperplasia of hepatic progenitor cells, as well as benign and malignant tumors that display both phenotypic and genotypic resemblances to human counterparts. The results reveal MET to be a crucial switch in the development of the liver; dramatize how different cellular compartments within a developmental lineage can give rise to distinctive tumor stem cells; delineate rules of tumor progression; provide evidence that the experimental tumors in mice are authentic models for human tumors; and support a role for MET in the genesis of human liver tumors. The models should be useful in elucidating the mechanisms of tumorigenesis and in the preclinical testing of new therapeutics.

Heterotopically transplanted hepatocyte survival depends on extracellular matrix components.

The novel approach of tissue engineering to treat many forms of liver diseases using hepatocytes requires sufficient numbers and sustained survival of the transplanted cells. It has been shown that providing extracellular matrix components extracted from Engelbreth-Holm-Swarm cells (EHS-ECMs) to heterotopically transplanted hepatocytes allows significantly greater hepatocyte survival. We investigated the survival and morphology of hepatocytes and EHS-ECMs transplanted under the kidney capsule compared with hepatocytes with growth factor-reduced EHS-ECMs in mice. Both the EHS-ECMs and growth factor-reduced EHS-ECMs showed a large number of surviving hepatocytes under the kidney capsule without any intergroup differences. Histologically, transplanted hepatocytes in both groups retained their characteristic morphologies and formed small liver tissues. These data indicate that extracellular matrix components are the predominant factor in EHS-ECMs required to maintain hepatocytes at heterotopic sites.

Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo.

We previously demonstrated that sustainable enhanced levels of transgene products could be expressed from a bacterial DNA-free expression cassette either formed from a fragmented plasmid in mouse liver or delivered as a minicircle vector. This suggested that bacterial DNA sequences played a role in episomal transgene silencing. To further understand the silencing mechanism, we systematically altered the DNA components in both the expression cassette and the bacterial backbone, and compared the gene expression profiles from mice receiving different DNA forms. In nine vectors tested, animals that received the purified expression cassette alone always expressed persistently higher levels of transgene compared to 2fDNA groups. In contrast, animals that received linearized DNA by a single cut in the bacterial backbone had similar expression profiles to that of intact plasmid groups. All three linear DNAs formed large concatemers and small circles in mouse liver, while ccDNA remained intact. In all groups, the relative amount of vector DNA in liver remained similar. Together, these results further established that the DNA silencing effect was mediated by a covalent linkage of the expression cassette and the bacteria DNA elements.

From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy.

Gene transfer vectors based on the human adeno-associated virus serotype 2 (AAV-2) have been developed and tested in pre-clinical studies for almost 20 years, and are currently being evaluated in clinical trials. So far, all these studies have provided evidence that AAV-2 vectors possess many properties making them very attractive for therapeutic gene delivery to humans, such as a lack of pathogenicity or toxicity, and the ability to confer long-term gene expression. However, there is concern that two restrictions of AAV-2 vectors might limit their clinical use in humans. First, these vectors are rather inefficient at transducing some cells of therapeutic interest, such as liver and muscle cells. Second, gene transfer might be hampered by neutralizing anti-AAV-2 antibodies, which are highly prevalent in the human population. In efforts to overcome both limitations, an increasing number of researchers are now focusing on the seven other naturally occurring serotypes of AAV (AAV-1 and AAV-3 to -8), which are structurally and functionally different from AAV-2. To this end, several strategies have been devised to cross-package an AAV-2 vector genome into the capsids of the other AAV serotypes, resulting in a new generation of "pseudotyped" AAV vectors. In vitro and in vivo, these novel vectors were shown to have a host range different from AAV-2, and to escape the anti-AAV-2 immune response, thus underscoring the great potential of this approach. Here the biology of the eight AAV serotypes is summarized, existing technology for pseudotyped AAV vector production is described, initial results from pre-clinical evaluation of the vectors are reviewed, and finally, the prospects of these promising novel tools for human gene therapy are discussed.

Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer.

Sustainable correction of severe human genetic disorders of self-renewing tissues, such as the blistering skin disease junctional epidermolysis bullosa (JEB), is facilitated by stable genomic integration of therapeutic genes into somatic tissue stem cells. While integrating viral vectors can achieve this, they suffer from logistical and biosafety concerns. To circumvent these limitations, we used the Sleeping Beauty transposable element to integrate the LAMB3 cDNA into genomes of epidermal holoclones from six unrelated JEB patients. These cells regenerate human JEB skin that is normalized at the level of laminin 5 protein expression, hemidesmosome formation and blistering. Transposon-mediated gene delivery therefore affords an opportunity for stable gene delivery in JEB and other human diseases.

Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males.

A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.

Hepatocyte transplantation: clinical and experimental application.

Hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency. Based on a significant body of work, the technique of hepatocyte transplantation has recently moved into the clinic in order to reestablish liver function without organ transplantation or to bridge the time between whole-organ liver transplantation. In addition, hepatocyte transplantation has also been proposed as a liver-directed gene therapy for a number of inherited hepatic disorders by transplanting either freshly isolated hepatocytes or genetically altered hepatocytes. To establish a research system based on the developing technology of hepatocyte transplantation, chimeric small animal models using human hepatocytes have recently been established, which would allow the study of human hepatocyte-specific functions, such as hepatitis viral infection and replication in vivo. Various aspects related to the recent progress and existing obstacles in the area of hepatocyte transplantation are summarized in this report.

Approaches for generating recombinant adenovirus vectors.

Various methods have been developed to facilitate the generation of recombinant adenovirus vectors, and three commercially available methods have been most widely used: the homologous recombination method in E1-complement cell lines, the homologous recombination method in bacteria, and an in vitro ligation method based on simple routine plasmid construction. These methods can insert foreign genes not only into the E1 deletion region, but also into the E3 deletion region, thereby permitting the construction of a binary transgene expression system in which heterologous genes can be inserted into both the E1 and E3 regions. By modifying the latter two methods, fiber-mutant adenovirus vectors can be also constructed in order to modify vector tropism. In this paper, we review recent advances in the construction of first generation adenovirus vectors and fiber-modified adenovirus vectors.

Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk.

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

Modified HIV-1 based lentiviral vectors have an effect on viral transduction efficiency and gene expression in vitro and in vivo.

Gene transfer using lentiviral vectors has been recently shown to be enhanced with cis-acting elements in a cell-type-dependent manner in vivo. For this reason, the study reported here was designed to modify lentiviral vectors that express lacZ, human factor IX (FIX), or human alpha1-anti-trypsin (AAT) to study the effect of different cis DNA elements on transduction efficiencies. We found that incorporation of the central polypurine tract sequence (cppt) increased transduction efficiency in vitro while increasing the transduction of non-cell-cycling hepatocytes in vivo. C57Bl/6 scid mice that were administered lentiviral vectors devoid of the cppt (2 x 10(8) transducing units (T.U.)/mouse) had 81% of their lacZ-transduced hepatocytes colabeled with the cell cycle marker 5'-bromo-2'-deoxyuridine (BrdU). In contrast, inclusion of the cppt reduced the colabeling in mouse hepatocytes by 50%. Further modifications in the lentiviral vectors were performed to enhance viral titer and gene expression. We found that the inclusion of a matrix attachment region (MAR) from immunoglobulin-kappa (Igkappa) significantly increased the transduction efficiency, as measured by transgene protein expression and proviral DNA copy number, compared with vectors without Igkappa MAR. In vitro studies using human hepatoma cells demonstrated a significant increase (two- to fourfold) in human AAT and human FIX production when the Igkappa MAR was incorporated. In vivo transduction of partially hepatectomized C57Bl/6 mice given an optimized lentiviral vector containing the cppt and Igkappa MAR (2 x 10(8) T.U./mouse) resulted in sustained therapeutic levels of serum FIX (approximately 65 ng/ml). Our study demonstrates the importance of cis-acting elements to enhancing the transduction ability of lentiviral vectors and the expression of vector transgenes.

Epstein-Barr virus/human vector provides high-level, long-term expression of alpha1-antitrypsin in mice.

We have constructed plasmid DNA vectors that contain Epstein-Barr virus (EBV) sequences and the human gene (SERPINA1) encoding alpha1-Antitrypsin (AAT). We demonstrate that a plasmid carrying the full SERPINA1 on a 19-kb genomic fragment and the EBV gene EBNA1 and its family of repeats binding sites undergoes efficient extrachromosomal replication in dividing mammalian tissue culture cells. Therefore, use of a whole genomic therapeutic gene to provide both replication and gene expression may be an effective gene therapy vector design, if the target cells are dividing. The efficacy of this same vector for expression of AAT in vivo in the nondividing cells of mouse liver was determined by hydrodynamic injection of naked plasmid DNA by means of the tail vein. A single injection of an EBV/genomic SERPINA1 vector provided >300 microg/ml of AAT, which approached normal plasma levels and persisted for the >9-month duration of the experiment. These data exceed most previously reported values, probably due to sequences in the genomic DNA that resist silencing of gene expression, possibly in combination with favorable effects on expression provided by the EBV sequences. These results demonstrate that plasmid DNA with the correct cis-acting sequences can provide in vivo long-term expression of protein at high levels that are therapeutically relevant for gene therapy.

Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo.

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10(11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than approximately 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.