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Posts Tagged ‘gene modification’


CD-4 Therapy for Solid Tumors

Curator: Larry H. Bernstein, MD, FCAP

 

CD4 T-cell Immunotherapy Shows Activity in Solid Tumors

Alexander M. Castellino, PhD

http://www.medscape.com/viewarticle/862095

For the first time, treatment with genetically engineered T-cells has used CD4 T-cells instead of the CD8 T-cells, which are used in the chimeric antigen receptor (CAR) T-cell approach. Early data suggest that this CD4 T-cell approach has activity against solid tumors, whereas the CAR T-cell approach so far has achieved dramatic success in hematologic malignancies.

In the new approach, CD4 T-cells were genetically engineered to target MAGE-A3, a protein found on many tumor cells. The treatment was found to be safe in patients with metastatic cancers, according to data from a phase 1 clinical study presented here at the American Association for Cancer Research (AACR) 2016 Annual Meeting.

“This is the first trial testing an immunotherapy using genetically engineered CD4 T-cells,” senior author Steven A. Rosenberg, MD, PhD, chief of the Surgery Branch at the National Cancer Institute (NCI), told Medscape Medical News.

Most approaches use CD8 T-cells. Although CD8 T-cells are known be cytotoxic and CD4 T-cells are normally considered helper cells, CD4 T-cells can induce tumor regression, he said.

Louis M. Weiner, MD, director of the Lombardi Comprehensive Cancer Center at Georgetown University, in Washington, DC, indicated that in contrast with CAR T-cells, these CD4 T-cells target proteins on solid tumors. “CAR T-cells are not tumor specific and do not target solid tumors,” he said.

Engineering CD4 Cells

Immunotherapy with engineered CD4 T-cells was personalized for each patient whose tumors had not responded to or had recurred following treatment with least one standard therapy. The immunotherapy was specific for patients in whom a specific human leukocyte antigen (HLA) — HLA-DPB1*0401 — was found to be expressed on their cells and whose tumors expressed MAGE-A3.

MAGE-A3 belongs to a class of proteins expressed during fetal development. The expression is lost in normal adult tissue but is reexpressed on tumor cells, explained presenter Yong-Chen William Lu, PhD, a research fellow in the Surgery Branch of the NCI.

Targeting MAGE-A3 is relevant, because it is frequently expressed in a variety of cancers, such as melanoma and urothelial, esophageal, and cervical cancers, he pointed out.

 Researchers purified CD4 T-cells from the peripheral blood of patients. Next, the CD4 T-cells were genetically engineered with a retrovirus carrying the T-cell receptor (TCR) gene that recognizes MAGE-A3. The modified cells were grown ex vivo and were transferred back into the patient.

Clinical Results

Dr Lu presented data for 14 patients enrolled into the study: eight patients received cell doses from 10 million to 30 billion cells, and six patients received up to 100 billion cells.

This was similar to a phase 1 dose-finding study, except the researchers were seeking to determine the maximum number of genetically engineered CD4 T-cells that a patient could safely receive.

One patient with metastatic cervical cancer, another with metastatic esophageal cancer, and a third with metastatic urothelial cancer experienced partial objective responses. At 15 months, the response is ongoing in the patient with cervical cancer; after 7 months of treatment, the response was durable in the patient with urothelial cancer; and a response lasting 4 months was reported for the patient with esophageal cancer.

Dr Lu said that a phase 2 trial has been initiated to study the clinical responses of this T-cell receptor therapy in different types of metastatic cancers.

In his discussion of the paper, Michel Sadelain, MD, of the Memorial Sloan Kettering Cancer Center, New York City, said, “Although therapy with CD4 cells has been evaluated using endogenous receptor, this is the first study using genetically engineered CD4 T-cells.”

Although the study showed that therapy with genetically engineered T-cells is safe and efficacious at least in three patients, the mechanism of cytotoxicity remains unclear, Dr Sadelain indicated.

Comparison With CAR T-cells

CAR T-cells act in much the same way. CARs are chimeric antigen receptors that have an antigen-recognition domain of an antibody (the V region) and a “business end,” which activates T-cells. In this case, CD8 T-cells from the patients are used to genetically engineer T-cells ex vivo. In the majority of cases, dramatic responses have been seen in hematologic malignancies.

CARs, directed against self-proteins, result in on-target, off-tumor effects, Gregory L. Beatty, MD, PhD, assistant professor of medicine at the University of Pennsylvania, in Philadelphia, indicated when he reported the first success story of CAR T-cells in a solid pancreatic cancer tumor.

Side effects of therapy with CD4 T-cells targeting MAGE-A3 were different and similar to side effects of chemotherapy, because patients received a lymphodepleting regimen of cyclophosphamide and fludabarine. Toxicities included high fever, which was experienced by the majority of patients (12/14). The fever lasted 1 to 2 weeks and was easily manageable.

High levels of the cytokine interleukin-6 (IL-6) were detected in the serum of all patients after treatment. However, the elevation in IL-6 levels was not considered to be a cytokine release syndrome, because no side effects occurred that correlated with the syndrome, Dr Liu indicated.

He also indicated that future studies are planned that will employ genetically engineered CD4 T-cells in combination with programmed cell death protein 1–blocking antibodies.

This study was funded by Intramural Research Program of the National Institutes of Health. The NCI’s research and development of T-cell receptor therapy targeting MAGE-A3 are supported in part under a cooperative research and development agreement between the NCI and Kite Pharma, Inc. Kite has an exclusive, worldwide license with the NIH for intellectual property relating to retrovirally transduced HLA-DPB1*0401 and HLA A1 T-cell receptor therapy targeting MAGE-A3 antigen. Dr Lu and Dr Rosenberg have disclosed no relevant financial relationships.

American Association for Cancer Research (AACR) 2016 Annual Meeting: Abstract CT003, presented April 17, 2016.

 

Searches Related to immunotherapy using genetically engineered CD4 T-cells

 

Genetic engineering of T cells for adoptive immunotherapy

To be effective for the treatment of cancer and infectious diseases, T cell adoptive immunotherapy requires large numbers of cells with abundant proliferative reserves and intact effector functions. We are achieving these goals using a gene therapy strategy wherein the desired characteristics are introduced into a starting cell population, primarily by high efficiency lentiviral vector-mediated transduction. Modified cells are then expanded using ex vivo expansion protocols designed to minimally alter the desired cellular phenotype. In this article, we focus on strategies to (1) dissect the signals controlling T cell proliferation; (2) render CD4 T cells resistant to HIV-1 infection; and (3) redirect CD8 T cell antigen specificity.
Adoptive T cell therapy is a form of transfusion therapy involving the infusion of large numbers of T cells with the aim of eliminating, or at least controlling, malignancies or infectious diseases. Successful applications of this technique include the infusion of CMV-or EBVspecific CTLs to protect immunosuppressed patients from these transplantation-associated diseases [1,2]. Furthermore, donor lymphocyte infusions of ex vivo-expanded allogeneic T cells have been used to successfully treat hematological malignancies in patients with relapsed disease following allogeneic hematopoietic stem cell transplant [3]. However, in many other malignancies and chronic viral infections such as HIV-1, adoptive T cell therapy has achieved inconsistent and/or marginal successes. Nevertheless, there are compelling reasons for optimism on this strategy. For example, the existence of HIV-positive elite non-progressors [4], as well as the correlation between the presence of intratumoral T cells and a favorable prognosis in malignancies such as ovarian [5,6] and colon carcinoma [7,8], provides in vivo evidence for the critical role of the immune system in controlling both HIV and cancer.
The key to successful adoptive immunotherapy strategies appears to consist of (1) using the “right” T cell type(s) and (2) obtaining therapeutically effective numbers of these cells without compromising their effector functions or their ability to engraft within the host. This article is focused on strategies employed in our laboratory to generate the “right” cell through genetic engineering approaches, with an emphasis on redirecting the antigen specificity of CD8 T cells, and rendering CD4 T cells resistant to HIV-1 infection. The article by Paulos et al. describes the evolving process of how to best obtain therapeutically effective numbers of the “right” cells by optimizing ex vivo cell expansion strategies.
Our laboratory’s overall strategy and flow plan for development and evaluation of engineered T cells is depicted in Fig. 1. We work almost exclusively with primary human T cells; little or no work is performed with conventional established cell lines. Thus, we benefit substantially from our close association with the UPenn Human Immunology Core. The Core performs leukaphereses on healthy donors 2–3 times a week, and provides purified peripheral blood mononuclear cell subsets, ensuring a constant influx of fresh human T cells into our laboratory. We have extensive experience in developing both bead- and cell-based artificial antigen presenting cells (aAPCs), as described in detail in the article by Paulos et al. The ability to genetically modify T cells at high efficiency is critical for virtually every project within the laboratory. We have adapted the lentiviral vector system described by Dull [15] for most, but not all, of the engineering applications in our laboratory.
CD4 T cells are the primary target of HIV-1, and decreasing CD4 T cell numbers is a hallmark of advancing HIV-1 disease [34]. Thus, strategies that protect CD4 T cells from HIV-1 infection in vivo would conceivably provide sufficient immunological help to control HIV-1 infection. Our early observations that CD3/CD28 costimulation resulted in improved ex vivo expansion of CD4 T cells from both healthy and HIV-infected donors, as well as enhanced resistance to HIV-1 infection [35,36], ultimately led to the first-in-human trial of lentiviral vector-modified CD4 T cells [37]. In this trial, CD4 T cells from HIV-positive subjects who had failed antiretroviral therapy were transduced with a lentiviral vector encoding an antisense RNA that targeted a 937 bp region in the HIV-1 envelope gene. Preclinical studies demonstrated that this antisense region, directed against the HIV-1NL4-3 envelope, provided robust protection from a broad range of both R5-and X4-tropic HIV-1 isolates [38]. One year after administration of a single dose of the gene-modified cells, four of the five enrolled patients had increased peripheral blood CD4 T cell counts, and in one subject, a 1.7 log decrease in viral load was observed. Finally, in two of the five patients, persistence of the gene-modified cells was detected one year post-infusion.
Since its identification as the primary co-receptor involved in HIV transmission, CCR5 has attracted considerable attention as a target for HIV therapy [42,43]. Indeed, “experiments of nature” have shown that individuals with a homozygous CCR5 Δ32 deletion are highly resistant to HIV-1 infection. Thus, we hypothesized that knocking out the CCR5 locus would generate CD4 T cells permanently resistant to infection by R5 isolates of HIV-1. To test this hypothesis we took advantage of zinc-finger nuclease (ZFN) technology [44]. ZFNs introduce sequencespecific double-strand DNA breakage, which is imperfectly repaired by non-homologous endjoining. This results in the permanent disruption of the genomic target, a process termed genome editing (Fig. 3).
Genetic modification of T cells to redirect antigen specificity is an attractive strategy compared to the lengthy process of growing T cell lines or CTL clones for adoptive transfer. Genetically modified, adoptively transferred T cells are capable of long-term persistence in humans [37, 46,47], demonstrating the feasibility of this approach. When compared to the months it can take to generate an infusion dose of antigen-specific CTL lines or clones from a patient, a homogeneous population of redirected antigen-specific cells can be expanded to therapeutically relevant numbers in about two weeks [3]. Several strategies are being explored to bypass the need to expand antigen-specific T cells for adoptive T cell therapy. The approaches currently studied in our laboratory involve the genetic transfer of chimeric antigen receptors and supraphysiologic T cell receptors.
Chimeric antigen receptors (CARs or T-bodies) are artificial T cell receptors that combine the extracellular single-chain variable fragment (scFv) of an antibody with intracellular signaling domains, such as CD3ζ or Fc(ε)RIγ [48–50]. When expressed on T cells, the receptor bypasses the need for antigen presentation on MHC since the scFv binds directly to cell surface antigens. This is an important feature, since many tumors and virus-infected cells downregulate MHCI, rendering them invisible to the adaptive immune system. The high-affinity nature of the scFv domain makes these engineered T cells highly sensitive to low antigen densities. In addition, new chimeric antigen receptors are relatively easy to produce from hybridomas. The key to this approach is the identification of antigens with high surface expression on tumor cells, but reduced or absent expression on normal tissues.  Since one can redirect both CD4 and CD8 T cells, the T-body approach to immunotherapy represents a near universal “off the shelf” method to generate large numbers of antigen-specific helper and cytotoxic T cells.
Many T-bodies targeting diverse tumors have been developed [51], and four have been evaluated clinically [52–55]. Three of the four studies were characterized by poor transgene expression and limited T-body engraftment. However, in a study of metastatic renal cell carcinoma using a T-body directed against carbonic anhydrase IX [55], T-body-expressing cells were detectable in the peripheral blood for nearly 2 months post-administration.
The major goals in the T-body field currently are to optimize their engraftment and maximize their effector functions. Our laboratory is addressing both problems simultaneously through an in-depth study of the requirements for T-body activation. We hypothesize that their limited persistence is due to incomplete cell activation due to the lack of costimulation. While naïve T cells depend on costimulation through CD28 ligation to avoid anergy and undergo full activation in response to antigen, it is recognized that effector cells also require costimulation to properly proliferate and produce cytokines [56]. Previous studies have shown that providing CD28 costimulation is crucial for the antitumoral function of adoptively transferred T cells and T-bodies [57–59]. Unlike conventional T cell activation, which requires two discrete signals, T-bodies can be engineered to provide both costimulation and CD3 signaling through one binding event.
A different approach for redirecting specificity to T cells for adoptive immunotherapy involves the genetic transfer of full-length TCR genes. A T cell’s specificity for its cognate antigen is solely determined by its TCR. Genes encoding the α and β chains of a T cell receptor (TCR) can be isolated from a T cell specific for the antigen of interest and restricted to a defined HLA allele, inserted into a vector, and then introduced into large numbers of T cells of individual patients that share the restricting HLA allele as well as the targeted antigen. In 1999, Clay and colleagues from Rosenberg’s group at the National Cancer Institute were the first to report the transfer of TCR genes via a retroviral vector into human lymphocytes and to show that T cells gained stable reactivity to MART-1 [67]. To date, many others have shown that the same approach can be used to transfer specificity for multiple viral and tumor associated antigens in mice and human systems. These T cells gain effector functions against the transferred TCR’s cognate antigen, as defined by proliferation, cytokine production, lysis of targets presenting the antigen, trafficking to tumor sites in vivo, and clearance of tumors and viral infection.
In 2006, Rosenberg’s group redirected patients’ PBLs with the naturally occurring, MART-1- specific TCR reported in 1999 by Clay. In the first clinical trial to test TCR-transfer immunotherapy, these modified T cells were infused into melanoma patients [68]. While the transduced T cells persisted in vivo, only two of the 17 patients had an objective response to this therapy. One issue revealed by the study was the poor expression of the transgenic TCRs by the transferred T cells. Nonetheless, the results from this trial showed the potential of TCR transfer immunotherapy as a safe form of therapy for cancer and highlighted the need to optimize such therapy to attain maximum potency.
The adoptive immunotherapy field is advancing by a tried-and-true method: learning from disappointments and moving forward. Our ability to fully realize the therapeutic potential of adoptive T cell therapy is tied to a more complete understanding of how human T cells receive signals, kill targets, and modulate effective immune responses. Our goal is to perform labbased experiments that provide insight into how primary T cells function in a manner that will facilitate and enable adoptive T cell therapy clinical trials. Our ability to efficiently modify (and expand) T cells ex vivo provides the opportunity to deliver sufficient immune firepower where it has heretofore been lacking. Sustained transgene expression, coupled with enhanced in vivo engraftment capability, will move adoptive immunotherapy into a realm where longterm therapeutic benefits are the norm rather than the exception.
Genetic Modification of T Lymphocytes for Adoptive Immunotherapy

Claudia Rossig1 and Malcolm K. Brenner2
Molecular Therapy (2004) 10, 5–18;   http://dx.doi.org:/10.1016/j.ymthe.2004.04.014      http://www.nature.com/mt/journal/v10/n1/full/mt20041193a.html

Adoptive transfer of T lymphocytes is a promising therapy for malignancies—particularly of the hemopoietic system—and for otherwise intractable viral diseases. Efforts to broaden the approach have been limited by the physiology of the T cells themselves and by a range of immune evasion mechanisms developed by tumor cells. In this review we show how genetic modification of T cells is being used preclinically and in patients to overcome these limitations, by incorporation of novel receptors, resistance mechanisms, and control genes. We also discuss how the increasing safety and effectiveness of gene transfer technologies will lead to an increase in the use of gene-modified T cells for the treatment of a wider range of disorders.

That gene transfer could be used to improve the effectiveness of T lymphocytes was apparent from the beginning of clinical studies in the field. T cells were the very first targets for genetic modification in human gene transfer experiments. Rosenberg’s group marked tumor-infiltrating lymphocytes ex vivo with a Moloney retroviral vector encoding neomycin phosphotransferase before reinfusing them and attempting to demonstrate selective accumulation at tumor sites. Shortly thereafter, Blaese and Anderson led a group that infused corrected T cells into two children with severe combined immunodeficiency due to ADA deficiency. While neither study was completely successful in terms of outcome, both showed the feasibility of ex vivo gene transfer into human cells and set the stage for many of the studies that followed. More recently, a second wave of interest in adoptive T cell therapies has developed, based on their success in the prevention and treatment of viral infections such as EBV and cytomegalovirus (CMV) and on their apparent ability to eradicate hematologic and perhaps solid malignancies1,2,3,4,5,6. There has been a corresponding increase in studies directed toward enhancing the antineoplastic and antiviral properties of the T cells. In this article we will review how gene transfer may be used to produce the desired improvements focusing on vectors and genes that have had clinical application.

Currently available viral and nonviral vector systems lack a pattern of biodistribution that would favor T cell transduction in vivo—as occurs, for example, with adenovectors and the liver or liposomal vectors and the lung. This lack of favorable biodistribution cannot yet be compensated for by the introduction of specific T-cell-targeting ligands into vectors. Hence, all T cell gene transfer studies conducted to date have used ex vivo transduction followed by adoptive transfer of gene-modified cells. This approach is inherently less attractive for commercial development than directin vivo gene transfer and has probably restricted interest in developing clinical applications using these cells. On the other hand, ex vivo transduction may be more readily controlled, characterized, and standardized than in vivo efforts and may ultimately produce a better defined final product (the transduced cell).

The gene products of suicide and coexpressed resistance genes are highly immunogenic and may induce immune-mediated rejection of the transduced cells. In one study, the persistence of adoptively transferred autologous CD8+ HIV-specific CTL clones modified to express the hygromycin phosphotransferase (Hy) gene and the herpesvirus thymidine kinase gene as a fusion gene was limited by the induction of a potent CD8+ class I MHC-restricted CTL response specific for epitopes derived from the Hy-tk protein126. Less immunogenic suicide and selection marker genes, preferably of human origin, may reduce the immunological inactivation of genetically modified donor lymphocytes. Human-derived prodrug-activating systems include the human folylpolyglutamate synthetase/methotrexate127, the deoxycytidine/cytosine arabinoside128, or the carboxylesterase/irinotecan129 systems. These systems do not activate nontoxic prodrugs but are based on enhancement of already potent chemotherapeutic agents. The administration of methotrexate to treat severe GVHD may not only kill transduced donor lymphocytes but may also have additional inhibitory activity on nontransduced but activated T cells.

Finally, endogenous proapoptotic molecules have been proposed as nonimmunogenic suicide genes. A chimeric protein that contains the FK506-binding protein FKBP12 linked to the intracellular domain of human Fas130 was recently introduced. Addition of the dimerizing prodrug induces Fas crosslinking with subsequent triggering of an apoptotic death signal.

Genetic engineering of T lymphocytes should help deliver on the promise of immunotherapies for cancer, infection, and autoimmune disease. Improvements in transduction, selection, and expansion techniques and the development of new viral vectors incapable of insertional mutagenesis will reduce the risks and further enhance the integration of T cell and gene therapies. Nonetheless, successful application of the proposed modifications to the clinical setting still requires many iterative studies to allow investigators to optimize the individual components of the approach.

Genetically modified T cells in cancer therapy: opportunities and challenges
Michaela Sharpe, Natalie Mount

 

The feasibility of T-cell adoptive transfer was first reported nearly 20 years ago (Walter et al., 1995) and the field of T-cell therapies is now poised for significant clinical advances. Recent clinical trial successes have been achieved through multiple small advances, improved understanding of immunology and emerging technologies. As the key challenges of T-cell avidity, persistence and ability to exert the desired anti-tumour effects as well as the identification of new target antigens are addressed, a broader clinical application of these therapies could be achieved. As the clinical data emerges, the challenge of making these therapies available to patients shifts to implementing robust, scalable and cost-effective manufacture and to the further evolution of the regulatory requirements to ensure an appropriate but proportionate system that is adapted to the characteristics of these innovative new medicines.

 

 

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Expanding the Genetic Alphabet and Linking the Genome to the Metabolome


English: The citric acid cycle, also known as ...

English: The citric acid cycle, also known as the tricarboxylic acid cycle (TCA cycle) or the Krebs cycle. Produced at WikiPathways. (Photo credit: Wikipedia)

Expanding the Genetic Alphabet and Linking the Genome to the Metabolome

 

Reporter& Curator:  Larry Bernstein, MD, FCAP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Unlocking the diversity of genomic expression within tumorigenesis and “tailoring” of therapeutic options

1. Reshaping the DNA landscape between diseases and within diseases by the linking of DNA to treatments

In the NEW York Times of 9/24,2012 Gina Kolata reports on four types of breast cancer and the reshaping of breast cancer DNA treatment based on the findings of the genetically distinct types, which each have common “cluster” features that are driving many cancers.  The discoveries were published online in the journal Nature on Sunday (9/23).  The study is considered the first comprehensive genetic analysis of breast cancer and called a roadmap to future breast cancer treatments.  I consider that if this is a landmark study in cancer genomics leading to personalized drug management of patients, it is also a fitting of the treatment to measurable “combinatorial feature sets” that tie into population biodiversity with respect to known conditions.   The researchers caution that it will take years to establish transformative treatments, and this is clearly because in the genetic types, there are subsets that have a bearing on treatment “tailoring”.   In addition, there is growing evidence that the Watson-Crick model of the gene is itself being modified by an expansion of the alphabet used to construct the DNA library, which itself will open opportunities to explain some of what has been considered junk DNA, and which may carry essential information with respect to metabolic pathways and pathway regulation.  The breast cancer study is tied to the  “Cancer Genome Atlas” Project, already reported.  It is expected that this work will tie into building maps of genetic changes in common cancers, such as, breast, colon, and lung.  What is not explicit I presume is a closely related concept, that the translational challenge is closely related to the suppression of key proteomic processes tied into manipulating the metabolome.

Saha S. Impact of evolutionary selection on functional regions: The imprint of evolutionary selection on ENCODE regulatory elements is manifested between species and within human populations. 9/12/2012. PharmaceuticalIntelligence.Wordpress.com

Hawrylycz MJ, Lein ES, Guillozet-Bongaarts AL, Shen EH, Ng L, et al. An anatomically comprehensive atlas of the adult human brain transcriptome. Nature  Sept 14-20, 2012

Sarkar A. Prediction of Nucleosome Positioning and Occupancy Using a Statistical Mechanics Model. 9/12/2012. PharmaceuticalIntelligence.WordPress.com

Heijden et al.   Connecting nucleosome positions with free energy landscapes. (Proc Natl Acad Sci U S A. 2012, Aug 20 [Epub ahead of print]).  http://www.ncbi.nlm.nih.gov/pubmed/22908247

2. Fiddling with an expanded genetic alphabet – greater flexibility in design of treatment (pharmaneogenesis?)

Diagram of DNA polymerase extending a DNA stra...

Diagram of DNA polymerase extending a DNA strand and proof-reading. (Photo credit: Wikipedia)

A clear indication of this emerging remodeling of the genetic alphabet is a new
study led by scientists at The Scripps Research Institute appeared in the
June 3, 2012 issue of Nature Chemical Biology that indicates the genetic code as
we know it may be expanded to include synthetic and unnatural sequence pairing (Study Suggests Expanding the Genetic Alphabet May Be Easier than Previously Thought, Genome). They infer that the genetic instructions for living organisms
that is composed of four bases (C, G, A and T)— is open to unnatural letters. An expanded “DNA alphabet” could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications. The implications of the application of this would further expand the translation of portions of DNA to new transciptional proteins that are heretofore unknown, but have metabolic relavence and therapeutic potential. The existence of such pairing in nature has been studied in Eukariotes for at least a decade, and may have a role in biodiversity. The investigators show how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases.  This could as well be translated into human diversity, and human diseases.

The Romesberg laboratory collaborated on the new study and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G). It had been clear that their chemical structures lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication, but that is not the case because other bonds can be in play.

The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, “intercalated” fashion that resembles a ‘mispair.’ In test after test, the NaM-5SICS pair was efficiently replicable even though it appeared that the DNA polymerase didn’t recognize it. Their structural data showed that the NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, “Watson-Crick” positioning when gripped by the polymerase during the crucial moments of DNA replication. NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures (like those found in oil) to be repelled by water molecules, and thus to cling together in a watery medium.

The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could be used to extend the DNA alphabet and that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been developed allowing for life based on other genetic systems.

3.  Studies that consider a DNA triplet model that includes one or more NATURAL nucleosides and looks closely allied to the formation of the disulfide bond and oxidation reduction reaction.

This independent work is being conducted based on a similar concep. John Berger, founder of Triplex DNA has commented on this. He emphasizes Sulfur as the most important element for understanding evolution of metabolic pathways in the human transcriptome. It is a combination of sulfur 34 and sulphur 32 ATMU. S34 is element 16 + flourine, while S32 is element 16 + phosphorous. The cysteine-cystine bond is the bridge and controller between inorganic chemistry (flourine) and organic chemistry (phosphorous). He uses a dual spelling, using  sulfphur to combine the two referring to the master catalyst of oxidation-reduction reactions. Various isotopic alleles (please note the duality principle which is natures most important pattern). Sulfphur is Methionine, S adenosylmethionine, cysteine, cystine, taurine, gluthionine, acetyl Coenzyme A, Biotin, Linoic acid, H2S, H2SO4, HSO3-, cytochromes, thioredoxin, ferredoxins, purple sulfphur anerobic bacteria prokaroytes, hydrocarbons, green sulfphur bacteria, garlic, penicillin and many antibiotics; hundreds of CSN drugs for parasites and fungi antagonists. These are but a few names which come to mind. It is at the heart of the Krebs cycle of oxidative phosphorylation, i.e. ATP. It is also a second pathway to purine metabolism and nucleic acids. It literally is the key enzymes between RNA and DNA, ie, SH thiol bond oxidized to SS (dna) cysteine through thioredoxins, ferredoxins, and nitrogenase. The immune system is founded upon sulfphur compounds and processes. Photosynthesis Fe4S4 to Fe2S3 absorbs the entire electromagnetic spectrum which is filtered by the Allen belt some 75 miles above earth. Look up chromatium vinosum or allochromatium species.  There is reasonable evidence it is the first symbiotic species of sulfphur anerobic bacteria (Fe4S4) with high potential mvolts which drives photosynthesis while making glucose with H2S.
He envisions a sulfphur control map to automate human metabolism with exact timing sequences, at specific three dimensional coordinates on Bravais crystalline lattices. He proposes adding the inosine-xanthosine family to the current 5 nucleotide genetic code. Finally, he adds, the expanded genetic code is populated with “synthetic nucleosides and nucleotides” with all kinds of customized functional side groups, which often reshape nature’s allosteric and physiochemical properties. The inosine family is nature’s natural evolutionary partner with the adenosine and guanosine families in purine synthesis de novo, salvage, and catabolic degradation. Inosine has three major enzymes (IMPDH1,2&3 for purine ring closure, HPGRT for purine salvage, and xanthine oxidase and xanthine dehydrogenase.

English: DNA replication or DNA synthesis is t...

English: DNA replication or DNA synthesis is the process of copying a double-stranded DNA molecule. This process is paramount to all life as we know it. (Photo credit: Wikipedia)

3. Nutritional regulation of gene expression,  an essential role of sulfur, and metabolic control 

Finally, the research carried out for decades by Yves Ingenbleek and the late Vernon Young warrants mention. According to their work, sulfur is again tagged as essential for health. Sulfur (S) is the seventh most abundant element measurable in human tissues and its provision is mainly insured by the intake of methionine (Met) found in plant and animal proteins. Met is endowed with unique functional properties as it controls the ribosomal initiation of protein syntheses, governs a myriad of major metabolic and catalytic activities and may be subjected to reversible redox processes contributing to safeguard protein integrity.

Consuming diets with inadequate amounts of methionine (Met) are characterized by overt or subclinical protein malnutrition, and it has serious morbid consequences. The result is reduction in size of their lean body mass (LBM), best identified by the serial measurement of plasma transthyretin (TTR), which is seen with unachieved replenishment (chronic malnutrition, strict veganism) or excessive losses (trauma, burns, inflammatory diseases).  This status is accompanied by a rise in homocysteine, and a concomitant fall in methionine.  The ratio of S to N is quite invariant, but dependent on source.  The S:N ratio is typical 1:20 for plant sources and 1:14.5 for animal protein sources.  The key enzyme involved with the control of Met in man is the enzyme cystathionine-b-synthase, which declines with inadequate dietary provision of S, and the loss is not compensated by cobalamine for CH3- transfer.

As a result of the disordered metabolic state from inadequate sulfur intake (the S:N ratio is lower in plants than in animals), the transsulfuration pathway is depressed at cystathionine-β-synthase (CβS) level triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and promoting its conversion to Met. They both stimulate comparable remethylation reactions from homocysteine (Hcy), indicating that Met homeostasis benefits from high metabolic priority. Maintenance of beneficial Met homeostasis is counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to CβS causing reducing molecules implicated in the regulation of the 3 desulfuration pathways

4. The effect on accretion of LBM of protein malnutrition and/or the inflammatory state: in closer focus

Hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly and liver production of  TTR integrates the dietary and stressful components of any disease spectrum. Thus we have a depletion of visceral transport proteins made by the liver and fat-free weight loss secondary to protein catabolism. This is most accurately reflected by TTR, which is a rapid turnover protein, but it is involved in transport and is essential for thyroid function (thyroxine-binding prealbumin) and tied to retinol-binding protein. Furthermore, protein accretion is dependent on a sulfonation reaction with 2 ATP.  Consequently, Kwashiorkor is associated with thyroid goiter, as the pituitary-thyroid axis is a major sulfonation target. With this in mind, it is not surprising why TTR is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequaled information on the alterations affecting overall protein nutritional status. Recent advances in TTR physiopathology emphasize the detecting power and preventive role played by the protein in hyper-homocysteinemic states.

Individuals submitted to N-restricted regimens are basically able to maintain N homeostasis until very late in the starvation processes. But the N balance study only provides an overall estimate of N gains and losses but fails to identify the tissue sites and specific interorgan fluxes involved. Using vastly improved methods the LBM has been measured in its components. The LBM of the reference man contains 98% of total body potassium (TBK) and the bulk of total body sulfur (TBS). TBK and TBS reach equal intracellular amounts (140 g each) and share distribution patterns (half in SM and half in the rest of cell mass). The body content of K and S largely exceeds that of magnesium (19 g), iron (4.2 g) and zinc (2.3 g).

TBN and TBK are highly correlated in healthy subjects and both parameters manifest an age-dependent curvilinear decline with an accelerated decrease after 65 years. Sulfur Methylation (SM) undergoes a 15% reduction in size per decade, an involutive process. The trend toward sarcopenia is more marked and rapid in elderly men than in elderly women decreasing strength and functional capacity. The downward SM slope may be somewhat prevented by physical training or accelerated by supranormal cytokine status as reported in apparently healthy aged persons suffering low-grade inflammation or in critically ill patients whose muscle mass undergoes proteolysis.

5.  The results of the events described are:

  • Declining generation of hydrogen sulfide (H2S) from enzymatic sources and in the non-enzymatic reduction of elemental S to H2S.
  • The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions.
  • Elemental S operates as co-factor of several (apo)enzymes critically involved in the control of oxidative processes.

Combination of protein and sulfur dietary deficiencies constitute a novel clinical entity threatening plant-eating population groups. They have a defective production of Cys, GSH and H2S reductants, explaining persistence of an oxidative burden.

6. The clinical entity increases the risk of developing:

  • cardiovascular diseases (CVD) and
  • stroke

in plant-eating populations regardless of Framingham criteria and vitamin-B status.
Met molecules supplied by dietary proteins are submitted to transmethylation processes resulting in the release of Hcy which:

  • either undergoes Hcy — Met RM pathways or
  • is committed to transsulfuration decay.

Impairment of CβS activity, as described in protein malnutrition, entails supranormal accumulation of Hcy in body fluids, stimulation of activity and maintenance of Met homeostasis. The data show that combined protein- and S-deficiencies work in concert to deplete Cys, GSH and H2S from their body reserves, hence impeding these reducing molecules to properly face the oxidative stress imposed by hyperhomocysteinemia.

Although unrecognized up to now, the nutritional disorder is one of the commonest worldwide, reaching top prevalence in populated regions of Southeastern Asia. Increased risk of hyperhomocysteinemia and oxidative stress may also affect individuals suffering from intestinal malabsorption or westernized communities having adopted vegan dietary lifestyles.

Ingenbleek Y. Hyperhomocysteinemia is a biomarker of sulfur-deficiency in human morbidities. Open Clin. Chem. J. 2009 ; 2 : 49-60.

7. The dysfunctional metabolism in transitional cell transformation

A third development is also important and possibly related. The transition a cell goes through in becoming cancerous tends to be driven by changes to the cell’s DNA. But that is not the whole story. Large-scale techniques to the study of metabolic processes going on in cancer cells is being carried out at Oxford, UK in collaboration with Japanese workers. This thread will extend our insight into the metabolome. Otto Warburg, the pioneer in respiration studies, pointed out in the early 1900s that most cancer cells get the energy they need predominantly through a high utilization of glucose with lower respiration (the metabolic process that breaks down glucose to release energy). It helps the cancer cells deal with the low oxygen levels that tend to be present in a tumor. The tissue reverts to a metabolic profile of anaerobiosis.  Studies of the genetic basis of cancer and dysfunctional metabolism in cancer cells are complementary. Tomoyoshi Soga’s large lab in Japan has been at the forefront of developing the technology for metabolomics research over the past couple of decades (metabolomics being the ugly-sounding term used to describe research that studies all metabolic processes at once, like genomics is the study of the entire genome).

Their results have led to the idea that some metabolic compounds, or metabolites, when they accumulate in cells, can cause changes to metabolic processes and set cells off on a path towards cancer. The collaborators have published a perspective article in the journal Frontiers in Molecular and Cellular Oncology that proposes fumarate as such an ‘oncometabolite’. Fumarate is a standard compound involved in cellular metabolism. The researchers summarize that shows how accumulation of fumarate when an enzyme goes wrong affects various biological pathways in the cell. It shifts the balance of metabolic processes and disrupts the cell in ways that could favor development of cancer.  This is of particular interest because “fumarate” is the intermediate in the TCA cycle that is converted to malate.

Animation of the structure of a section of DNA...

Animation of the structure of a section of DNA. The bases lie horizontally between the two spiraling strands. (Photo credit: Wikipedia)

The Keio group is able to label glucose or glutamine, basic biological sources of fuel for cells, and track the pathways cells use to burn up the fuel.  As these studies proceed, they could profile the metabolites in a cohort of tumor samples and matched normal tissue. This would produce a dataset of the concentrations of hundreds of different metabolites in each group. Statistical approaches could suggest which metabolic pathways were abnormal. These would then be the subject of experiments targeting the pathways to confirm the relationship between changed metabolism and uncontrolled growth of the cancer cells.

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