Homology-directed gene-editing approaches for hematopoietic stem and progenitor cell gene therapy

The advent of next-generation genome engineering tools like CRISPR-Cas9 has transformed the field of gene therapy, rendering targeted treatment for several incurable diseases. Hematopoietic stem and progenitor cells (HSPCs) continue to be the ideal target cells for gene manipulation due to their long-term repopulation potential. Among the gene manipulation strategies such as lentiviral gene augmentation, non-homologous end joining (NHEJ)-mediated gene editing, base editing and prime editing, only the homology-directed repair (HDR)-mediated gene editing provides the option of inserting a large transgene under its endogenous promoter or any desired locus. In addition, HDR-mediated gene editing can be applied for the gene knock-out, correction of point mutations and introduction of beneficial mutations. HSPC gene therapy studies involving lentiviral vectors and NHEJ-based gene-editing studies have exhibited substantial clinical progress. However, studies involving HDR-mediated HSPC gene editing have not yet progressed to the clinical testing. This suggests the existence of unique challenges in exploiting HDR pathway for HSPC gene therapy. Our review summarizes the mechanism, recent progresses, challenges, and the scope of HDR-based gene editing for the HSPC gene therapy.

Karyotype analysis of Aeluropus species (Poaceae)

Aeluropus, a member of Poaceae subfam. Chloridoideae, includes six species, three of which occur in Iran. They are perennial halophytes of deserts and coastal marshlands of Iran. The genus is considered as a rich genetic source for gene manipulation and using it for crop improvement. Previous studies showed that members of Chloridoideae have small chromosomes and the base chromosome number n = 10. There are few chromosome records for Aeluropus species. Somatic metaphases of seven populations of three Aeluropus species were studied. The first chromosome counts (2n = 20) based on Iranian material for three species, A. macrostachyus, A. littoralis and A. lagopoides, are concordant with previous records outside Iran; mitotic number for A. macrostachyus is recorded here for the first time.

Genetic engineering and gene manipulation: the right (of future generations) to surprise

Genetic engineering is currently at the forefront of biotechnological innovation and aspires to change once and for ever the way we understand and handle human nature. Especially the growth of Eugenics makes us visualise a different world, where humanity will not only dispense itself from the detrimental gene mutations that are accountable for fatal illnesses, but will also ameliorate through prenatal gene manipulation. In the first part of this paper, I will introduce the responsibility-oriented morality of Hans Jonas, who supports vividly all efforts of negative Eugenics but seems sceptical about genetic enhancement, since on the one hand we have no right to decide on behalf of our descendants on what is best for them, and on the other due to his view that the abundance of our genetic stock should not hang on parents’ desires. In the second part, I will correlate these oppositions with Hannah Arendt’s concept of “natality”. Not only do Jonas and Habermas invoke it with applause; “natality” also discloses the very essence of birth, namely that each newborn epitomises total unpredictability and promises to renew human affairs. So, gene manipulation for enhancement purposes seems to encroach on “natality’s” dominion and diminish future autonomy. Finally, I will argue that, if Arendt’s conceptual frame consolidates objections to positive Eugenics, each unborn child holds a right to surprise, the content of which is not limited to an individual level but touches society and humanity.

Delivery of Protein Kinase A by CRISPRMAX and Its Effects on Breast Cancer Stem-Like Properties

Protein kinase A (PKA) activation has recently been reported to inhibit epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) ability, which is considered to be responsible for chemoresistance and tumor recurrence in patients. While current studies mainly focus on gene manipulation of the EMT process, the direct delivery of PKA enzymes to cancer cells has never been investigated. Here, we utilize the commercial Lipofectamine CRISPRMAX reagent to directly deliver PKAs to breast cancer cells and evaluate its effects on EMT regulation. We optimized the delivery parameters with fluorescent-labeled bovine serum albumin, and successfully delivered fluorescent PKAs through CRISPRMAX into breast cancer cells. Then, we evaluated the biological effects by immunofluorescence, flow cytometry, mammosphere assay, and chemoresistance assay. Our data showed the expression of EMT-related markers, α-smooth muscle actin and N-cadherin, was downregulated after CRISPRMAX-PKA treatment. Although the CD44+/CD24− population did not change considerably, the size of mammospheres significantly decreased. In paclitaxel and doxorubicin chemoresistance assays, we noticed PKA delivery significantly inhibited paclitaxel resistance rather than doxorubicin resistance. Taken together, these results suggest our direct enzyme delivery can be a potential strategy for inhibiting EMT/CSC-associated traits, providing a safer approach and having more clinical translational efficacy than gene manipulation. This strategy will also facilitate the direct testing of other target enzymes/proteins on their biological functions.

In ovo electroporation of chicken limb bud ectoderm

Deciphering how ectodermal tissues form, and how they maintain their integrity, is crucial for understanding epidermal development and pathogenesis. However, lack of simple and rapid gene manipulation techniques limits genetic studies to elucidate mechanisms underlying these events. Here we describe have an easy method for electroporation of chick embryo limb bud ectoderm, enabling gene manipulation during ectoderm development and wound healing. Taking advantage of a small parafilm well that constrains DNA plasmids locally and the fact that the limb ectoderm arises from a defined site, we target the limb ectoderm forming region by in ovo electroporation. This approach results in efficient transgenesis of the limb ectodermal cells. Further, using a previously described Msx2 promoter, gene manipulation can be specifically targeted to the apical ectodermal ridge (AER), a signaling center regulating limb development. Using the electroporation technique to deliver a fluorescent marker into the embryonic limb ectoderm, we show its utility in performing time-lapse imaging during wound healing. This analysis revealed previously unrecognized dynamic remodeling of the actin cytoskeleton and lamellipodia formation at the edges of the wound. We find that the lamellipodia formation requires activity of Rac1 GTPase, suggesting its necessity for wound closure. Our method is simple and cheap, and permits high throughput tests for gene function during limb ectodermal development and wound healing.

Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts

Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial protoplasts/spheroplasts are used for enlargement because they lack cell wall. Though bacterial species that are capable of gene manipulation are limited, procedure for bacterial cell enlargement does not involve any gene manipulation technique. In order to prevent cell wall resynthesis during enlargement of protoplasts/spheroplasts, incubation media are supplemented with inhibitors of peptidoglycan biosynthesis such as penicillin. Moreover, metal ion composition in the incubation medium affects the properties of the plasma membrane. Therefore, in order to generate enlarged cells that are suitable for microinjection, metal ion composition in the medium should be considered. Experiment of bacterial protoplast or spheroplast enlargement is useful for studies on bacterial plasma membrane biosynthesis. In this paper, we have summarized the factors that influence bacterial cell enlargement.

Interferon: Role in health, current trends and therapeutic potentials

This mini review presents the current trends and topical issues on interferon. This article illustrates the theoretical background and details about interferon, its mechanism of action or roles in the prevention of microbial- or pathogenic disease progression, types and classes, and their therapeutic potentials. All the reported applications have shown that interferon has found its application in gene manipulation and expression of gene products; immunological techniques, viral disease treatment and in the treatment of cancers. Keywords: Interferon, cancers, cancer therapy, viral infection, viral disease treatment.