Treatment Of Human Genetic Diseases Research Articles

Revealing Differential RNA Editing Specificity of Human ADAR1 and ADAR2 in Schizosaccharomyces pombe.

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2's preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.

Advances in CRISPR/Cas gene therapy for inborn errors of immunity.

Inborn errors of immunity (IEIs) are a group of inherited disorders caused by mutations in the protein-coding genes involved in innate and/or adaptive immunity. Hematopoietic stem cell transplantation (HSCT) is a mainstay definitive therapy for many severe IEIs. However, the lack of HLA-matched donors increases the risk of developing severe immunological complications. Gene therapy provides long-term clinical benefits and could be an attractive therapeutic strategy for IEIs. In this review, we describe the development and evolution of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) gene-editing systems, including double-strand break (DSB)-based gene editing and DSB-free base editing or prime editing systems. Here, we discuss the advances in and issues associated with CRISPR/Cas gene editing tools and their potential as therapeutic alternatives for IEIs. We also highlight the progress of preclinical studies for the treatment of human genetic diseases, including IEIs, using CRISR/Cas and ongoing clinical trials based on this versatile technology.

Genome-Scale Analysis of Cellular Restriction Factors That Inhibit Transgene Expression from Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are one of the leading platforms for gene delivery for the treatment of human genetic diseases, but the antiviral cellular mechanisms that interfere with optimal transgene expression are incompletely understood. Here, we performed two genome-scale CRISPR screens to identify cellular factors that restrict transgene expression from recombinant AAV vectors. Our screens revealed several components linked to DNA damage response, chromatin remodeling, and transcriptional regulation. Inactivation of the Fanconi anemia gene FANCA; the human silencing hub (HUSH)-associated methyltransferase SETDB1; and the gyrase, Hsp90, histidine kinase, and MutL (GHKL)-type ATPase MORC3 led to increased transgene expression. Moreover, SETDB1 and MORC3 knockout improved transgene levels of several AAV serotypes as well as other viral vectors, such as lentivirus and adenovirus. Finally, we demonstrated that the inhibition of FANCA, SETDB1, or MORC3 also enhanced transgene expression in human primary cells, suggesting that they could be physiologically relevant pathways that restrict AAV transgene levels in therapeutic settings. IMPORTANCE Recombinant AAV (rAAV) vectors have been successfully developed for the treatment of genetic diseases. The therapeutic strategy often involves the replacement of a defective gene by the expression of a functional copy from the rAAV vector genome. However, cells possess antiviral mechanisms that recognize and silence foreign DNA elements thereby limiting transgene expression and its therapeutic effect. Here, we utilize a functional genomics approach to uncover a comprehensive set of cellular restriction factors that inhibit rAAV-based transgene expression. Genetic inactivation of selected restriction factors increased rAAV transgene expression. Hence, modulation of identified restriction factors has the potential to enhance AAV gene replacement therapies.

Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity.

The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential toward the treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on the incorporation of modifications into the single-guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. We developed a conjugation strategy that utilized bio-orthogonal chemistry to efficiently assemble functional sgRNAs containing nucleobase modifications. The described approach entails the chemical synthesis of two shorter RNA oligonucleotides: a 31-mer containing tetrazine (Tz) group and a 70-mer modified with a trans-cyclooctene (TCO) moiety. The two oligonucleotides were conjugated to form functional sgRNAs. The two-component conjugation methodology was utilized to synthesize a library of sgRNAs containing nucleobase modifications such as N1-methyladenosine (m1A), N6-methyladenosine (m6A), 2-thiouridine (s2U), and 4-thiouridine (s4U). The impact of these RNA modifications on overall CRISPR activity were investigated in vitro and in Cas9-expressing HEK293T cells.

Reconstructed glycosylase base editors GBE2.0 with enhanced C-to-G base editing efficiency and purity

Base editing techniques were developed for precise base conversion on cellular genomic DNA, which has great potential for the treatment of human genetic diseases. The glycosylase base editor (GBE) recently developed in our lab was used to perform C-to-G transversions in mammalian cells. To improve the application prospects of GBE, it is necessary to further increase its performance. With this aim, we replaced the human Ung in GBE with Ung1 from Saccharomyces cerevisiae. The resulting editor APOBEC-nCas9-Ung1 was tested at 17 chromosomal loci and was found to have an increased C-to-G editing efficiency ranging from 2.63% to 52.3%, with an average of 23.48%, which was a significant improvement over GBE, with an average efficiency of 15.54%, but with a decreased purity. For further improvement, we constructed APOBEC(R33A)-nCas9-Rad51-Ung1 with two beneficial modifications adapted from previous reports. This base editor was able to achieve even higher editing efficiency ranging from 8.70% to 72.1%, averaging 30.88%, while also exhibiting high C-to-G purity ranging from 35.57% to 92.92%, and was designated GBE2.0. GBE2.0 provides high C-to-G editing efficiency and purity in mammalian cells, making it a powerful genetic tool for scientific research or potential genetic therapies for disease-causing G/C mutations.

The Importance of the Epi-Transcriptome in Translation Fidelity.

RNA modifications play an essential role in determining RNA fate. Recent studies have revealed the effects of such modifications on all steps of RNA metabolism. These modifications range from the addition of simple groups, such as methyl groups, to the addition of highly complex structures, such as sugars. Their consequences for translation fidelity are not always well documented. Unlike the well-known m6A modification, they are thought to have direct effects on either the folding of the molecule or the ability of tRNAs to bind their codons. Here we describe how modifications found in tRNAs anticodon-loop, rRNA, and mRNA can affect translation fidelity, and how approaches based on direct manipulations of the level of RNA modification could potentially be used to modulate translation for the treatment of human genetic diseases.

Transcriptional Regulation of Autophagy: Mechanisms and Diseases.

Macro (Autophagy) is a catabolic process that relies on the cooperative function of two organelles: the lysosome and the autophagosome. The recent discovery of a transcriptional gene network that co-regulates the biogenesis and function of these two organelles, and the identification of transcription factors, miRNAs and epigenetic regulators of autophagy, demonstrated that this catabolic process is controlled by both transcriptional and post-transcriptional mechanisms. In this review article, we discuss the nuclear events that control autophagy, focusing particularly on the role of the MiT/TFE transcription factor family. In addition, we will discuss evidence suggesting that the transcriptional regulation of autophagy could be targeted for the treatment of human genetic diseases, such as lysosomal storage disorders (LSDs) and neurodegeneration.

A chemiluminescence biosensor based on the adsorption recognition function between Fe3O4@SiO2@GO polymers and DNA for ultrasensitive detection of DNA

In this work, a chemiluminescence (CL) biosensor was prepared for ultrasensitive determination of deoxyribonucleic acid (DNA) based on the adsorption recognition function between core-shell Fe3O4@SiO2 - graphene oxide (Fe3O4@SiO2@GO) polymers and DNA. The Fe3O4@SiO2@GO polymers were composed by GO and magnetite nanoparticles. And the core-shell polymers were confirmed by Scanning Electron Microscope (SEM), X-Ray Powder Diffraction (XRD) and Fourier Transform Infrared (FTIR). Then Fe3O4@SiO2@GO was modified by DNA. Based on the principle of complementary base, Fe3O4@SiO2@GO-DNA was introduced to the CL system and the selectivity, sensitivity of DNA detection was significantly improved. The adsorption properties of Fe3O4@SiO2@GO to DNA were researched through the adsorption equilibrium, adsorption kinetic and thermodynamics. Under optimized CL conditions, DNA could be assayed with the linear concentration range of 5.0×10−12–2.5×10−11mol/L. The detection limit was 1.7×10−12mol/L (3δ) and the relative standard deviation (RSD) was 3.1%. The biosensor was finally used for the determination of DNA in laboratory samples and recoveries ranged from 99% to 103%. The satisfactory results revealed the potential application of Fe3O4@SiO2@GO-DNA-CL biosensor in the diagnosis and the treatment of human genetic diseases.

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells.

We describe the development of ‘recCas9’, an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases.

Alternative Splicing Modification as a Treatment for Genetic Disorders

Alternative splicing is a co-transcriptional mechanism that regulates eukaryotic gene expression affecting the majority of human genes. In this mechanism, different sequences can be identified and removed from the pre-mRNA. Using alternatives splicing, multiple mRNA combinations of joined sequences can be produced from a single gene, increasing the coding potential of the genome. Malfunctions of alternative splicing events can affect the natural expression of different transcripts. Several strategies have been developed to regulate alternative splicing and the mechanisms underlying the functional and physiological implications of these tools are diverse. Collectively, these strategies are intended to improve the treatment of human genetic diseases.

Biosafety Considerations Using Gamma-Retroviral Vectors in Gene Therapy

Gene therapy has become a feasible and efficient strategy for the treatment of human genetic diseases. The main principle of a gene therapeutic regimen relies on the delivery of a corrected gene of interest in human cells. In about one fifth of the clinical trials, gamma-retroviral vectors are used as gene-transfer vehicle. However, previous successful gene therapy trials revealed gamma-retroviral vector-mediated severe adverse events: Upregulation of proto-oncogenes led to malignant transformation of the affected cells and tumor progression. These severe adverse events enhanced the development of new 'safer' gamma-retroviral vectors and comprehensive biosafety studies. This review highlights all possible safety and biosafety risks of gamma-retroviral vectors.

Studies on Nonsense Mediated Decay Reveal Novel Therapeutic Options for Genetic Diseases

Scientific breakthroughs have often led to commercially viable patents mainly in the field of engineering. Commercialization in the field of medicine has been restricted mostly to machinery and engineering on the one hand and therapeutic drugs for common chronic ailments such as cough, cold, headache, etc, on the other. Sequencing of the human genome has attracted the attention of pharmaceutical companies and now biotechnology has become a goldmine for commercialization of products and processes. Recent advances in our understanding of basic biological processes have resulted in the opening of new avenues for treatment of human genetic diseases, especially single gene disorders. A significant proportion of human genetic disorders have been shown to be caused due to degradation of transcripts for specific genes through a process called nonsense mediated decay (NMD). The modulation of NMD provides a viable therapeutic option for treatment of several genetic disorders and therefore has been a good prospect for patenting and commercialization. In this review the molecular basis for NMD and attempts to treat genetic diseases which result from NMD are discussed.

ChemInform Abstract: Designer Aminoglycosides: The Race to Develop Improved Antibiotics and Compounds for the Treatment of Human Genetic Diseases.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Designer aminoglycosides: the race to develop improved antibiotics and compounds for the treatment of human genetic diseases

Aminoglycosides are highly potent, broad-spectrum antibiotics that exert their bactericidal therapeutic effect by selectively binding to the decoding aminoacyl site (A-site) of the bacterial 16 S rRNA, thereby interfering with translational fidelity during protein synthesis. The appearance of bacterial strains resistant to these drugs, as well as their relative toxicity, have inspired extensive searches towards the goal of obtaining novel molecular designs with improved antibacterial activity and reduced toxicity. In the last few years, a new, aminoglycoside dependent therapeutic approach for the treatment of certain human genetic diseases has been identified. These treatments rely on the ability of certain aminoglycosides to induce mammalian ribosomes to readthrough premature stop codon mutations. This new and challenging task has introduced fresh research avenues in the field of aminoglycoside research. Recent observations and current challenges in the design of aminoglycosides with improved antibacterial activity and the treatment of human genetic diseases are discussed.

Differential Selectivity of Natural and Synthetic Aminoglycosides towards the Eukaryotic and Prokaryotic Decoding A Sites

The lack of absolute prokaryotic selectivity of natural antibiotics is widespread and is a significant clinical problem. The use of this disadvantage of aminoglycoside antibiotics for the possible treatment of human genetic diseases is extremely challenging. Here, we have used a combination of biochemical and structural analysis to compare and contrast the molecular mechanisms of action and the structure-activity relationships of a new synthetic aminoglycoside, NB33, and a structurally similar natural aminoglycoside apramycin. The data presented herein demonstrate the general molecular principles that determine the decreased selectivity of apramycin for the prokaryotic decoding site, and the increased selectivity of NB33 for the eukaryotic decoding site. These results are therefore extremely beneficial for further research on both the design of new aminoglycoside-based antibiotics with diminished deleterious effects on humans, as well as the design of new aminoglycoside-based structures that selectively target the eukaryotic ribosome.

Redesign of aminoglycosides for treatment of human genetic diseases caused by premature stop mutations

A series of new derivatives of the clinically used aminoglycoside antibiotic paromomycin were designed, synthesized, and their ability to read-through premature stop codon mutations was examined in both in vitro translation system and ex vivo mammalian cultured cells. One of these structures, a pseudo-trisaccharide derivative, showed notably higher stop codon read-through activity in cultured cells compared to those of paromomycin and gentamicin.

Small molecule intervention in microtubule-associated human disease

Microtubules are essential for a number of cellular processes that include the transport of intracellular cargo or organelles across long distances and the assembly of the mitotic spindle. The identification of numerous microtubule-associated proteins and the progressive elucidation of the mechanisms of microtubule assembly and transport are beginning to have a profound impact on the study and treatment of human genetic disease. A number of seemingly unrelated phenotypes have now been linked to microtubular dysfunction, especially in systems dependent heavily on microtubule-based transport, such as neurons and ciliated cells. In parallel, the association of microtubule transport defects with human genetic disease has led to the realization that targeting various aspects of microtubular biology with small molecules might offer new therapeutic paradigms, including the development of new therapeutic utility for seemingly old drugs. In this review, we discuss the use of small molecules in the investigation of microtubule-associated processes and particularly the screens of chemical compound libraries for the identification of lead compounds with potential utility in microtubule-associated disease processes.

Identification and Characterization of Engrafted Human Cells in Human/Goat Xenogeneic Transplantation Chimerism

We have injected human CD34+lin- cells derived from cord blood (CB) into the goat fetuses via in utero at 45-55 days gestation under guidance of B-scan ultrasonograph. Sixty out of 68 fetuses injected survived to full term. The long-term survival of the human cells in transplant goat has been tested by various experimental methods, including FACS analysis, real-time PCR, RT-PCR, Southern-blot hybridization, FISH, as well as immunohistochemical assays. All the 60 transplant goats demonstrated engrafted human cells, including myeloid, B-lymphoid, and erythroid lineages. The yield of the human CD34+ cells varied, but was not linked with sex and age. High numbers of human cells could be detected for at least 16 months after birth. Immunohistochemical analyses revealed that the human cells were present not only in blood but also in other tissues, such as liver, of the transplant goats. In addition, a human-specific serum albumin and the hepatocyte nuclear factor (hHNF-3beta) mRNAs specific to human hepato-antigen could be readily detected in the livers of the transplant goats. Our results demonstrate that this in utero xenograft model should be useful for expansion of human HSC and possibly for the evaluating the effectiveness of prenatal treatment of human genetic diseases.

The Biotechnology Revolution: A Primer for Pharmacists

The Human Genome Project, an international effort, is ushering in an era of molecular medicine with newer and better approaches to the prevention, diagnosis, and treatment of human genetic diseases. Biotechnology, or genetic engineering, can be defined as the alteration of cells or biological molecules for specific applications. Biotechnology has contributed — and will contribute — to many major medical advances. To respond to the biotechnology revolution in health care, pharmacists must become familiar with this new field. With a state-of-the-art understanding of disease processes and drug effects, pharmacists will be ideally prepared to educate patients about disease states and the roles of old and new drugs in treatment plans.

Encapsidated adenovirus mini-chromosome-mediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration.

First (DeltaE1/E3) and second (DeltaE1+DeltaE2/E3/E4) generation adenovirus (Ad) vectors have been shown previously to be of limited use in the treatment of human genetic diseases due to the induction of a host cytotoxic T-cell mediated immune response against virally expressed genes. In addition, a limited cloning capacity of approximately 8 kb does not cater for the incorporation of large upstream sequences essential for regulated tissue-specific expression or inclusion of multiple gene-expression cassettes. In this study we have exploited our recently developed Ad-based vector, the encapsidated adenovirus mini-chromosome (EAM) from which all of the viral genes have been deleted. EAMs contain only the inverted terminal repeats required for replication and five cis -acting Ad encapsidation signals necessary for packaging. We have shown previously that EAMs can efficiently transduce a variety of cell types in vitro. In this study we demonstrate that EAMs can transduce and rescue cells from the neurosensory retina in vivo. EAM-mediated delivery of the beta subunit of cyclic GMP phosphodiesterase (PDE) cDNA to mice affected with retinal degeneration (rd) allows prolonged transgene expression and rescue of rod photoreceptor cells. RT-PCR analysis from the injected retina indicates that transgene products are present for at least 18 weeks post-injection. Both the alpha and beta subunits of PDE could be detected up to 90 days postnatal in EAM-injected rd retina by western analysis. A maximal PDE activity of 150 nm/min/mg was detected at 33 days postnatal. Examination of outer nuclear thickness showed significant differences up to 12 weeks post-injection. These results demonstrate an improved level of rescue over first-generation adenoviral vectors and suggest the possibility of successful EAM-mediated treatment of some retinal diseases in humans.