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T cell-mediated immune responses & signaling pathways activated by Toll-like Receptors

Posted in Cell Biology, Signaling & Cell Circuits, Clinical & Translational, Curation, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Gene Regulation, Human Immune System in Health and in Disease, tagged cell-mediated immunity, Toll-like receptor on September 7, 2015| Leave a Comment »

T cell-mediated immune responses & signaling pathways activated by Toll-like Receptors

Curator: Larry H. Bernstein, MD, FCAP

 

 

Series E. 2; 6.10

Bruce A. Beutler, Jules A. Hoffmann, Ralph M. Steinman

Bruce Beutler, MD

Regental Professor; Raymond and Ellen Willie Distinguished Chair in Cancer Research, in Honor of Laverne and Raymond Willie, Sr.

Department Center for Genetics of Host Defense, Immunology

Bruce Beutler, MD, the son of the “father of red cell metabolic pathways”, Ernest Beutler, was born in Chicago and grew up in Los Angeles, where Beutler did his groundbreaking work on hemolytic red cell disorders at City of Hope Hospital. He was infected with an interest in medicine and research at an early age, somewhat analagous to a comparison with Roger and Arthur Kornberg. He attended University of California San Diego (UCSD) Medical School,  where his interest in immunity was sparked by Abraham Braude, PhD, Professor Pathology and Medicine, and Chief of Microbiology.

Bruce Beutler, MD, discovered an important family of receptors that allow mammals to sense infections when they occur, triggering a powerful inflammatory response. For this work he received the 2011 Nobel Prize in Physiology or Medicine.

The period from 1950 to 1953 was critical to Abraham Braude’s career. During these years, Braude realized the importance of the microbiology laboratory not only in patient management, but also in physician education. He became convinced that infectious disease clinicians must not only be excellent microbiologists, but also exercise technical and administrative control of the diagnostic microbiology laboratory. He demonstrated and taught this principle of medical practice and education for the next 35 years as director of diagnostic microbiology and chief of infectious diseases at the Texas Southwestern Medical School  of the University of Texas from 1953-57, at the University of Pittsburgh from 1957 to 1969, and at the University of California at San Diego from 1967 until his death in December, 1984. He also was my mentor as a resident in 1972, and it was remarkable how he had developed his own antibiotic diffusion plates for sensitivity studies on bacterial isolates.

He discovered that the endotoxin of cold-growing bacteria that had contaminated refrigerated blood could cause vascular collapse and death of transfused people without bacterial multiplication in the body. This observation established the importance of endotoxin as the major virulence factor of Gram negative bacteria and set the stage for his later fundamental and applied research on the pathogenesis and treatment of life-threatening septic shock. This aspect of infection, in the host defense, is perhaps important in the development of the young Beutler.

During his years in Dallas, Pittsburgh, and San Diego, Braude made outstanding research contributions in three areas, anaerobic infections, pyelonephritis, and the endotoxin shock of Gram negative bacteremia. He devised a simple, effective method for culturing fastidious anaerobes, discovered Bacteroides penicillinase, and proved that so-called “sterile” brain abscesses are caused by anaerobes. His landmark papers on brain infections and sinusitis awakened us to the major role of anaerobes in infectious disease. His work on pyelonephritis ranged from devising realistic models of infection to elegant studies that helped explain the inadequacies of the immune responses of the kidney.

Beutler received his undergraduate degree from the University of California at San Diego in 1976, and his MD degree from the University of Chicago in 1981. After two years of residency at the University of Texas Southwestern Medical Center, he became a postdoctoral fellow and then an Assistant Professor at the Rockefeller University (1983-1986), where he isolated mouse tumor necrosis factor (TNF), and was the first to recognize TNF as a key executor of the inflammatory response. Returning to Dallas in 1986 as an HHMI investigator, he designed recombinant inhibitors of TNF that are widely used in the treatment of rheumatoid arthritis and other inflammatory diseases. He also used TNF as a biological endpoint in order to identify the receptor for bacterial lipopolysaccharide (LPS). This he achieved by positionally cloning the Lps mutation of mice, known to prevent all biological responses to LPS, including TNF production. He thus concluded that Toll-like receptor 4 (TLR4) acts as the signaling core of the LPS receptor and proposed that other TLRs might also recognize conserved molecular signatures of infection.

Moving in 2000 to the Scripps Research Institute, Beutler developed the largest mouse mutagenesis program in the world, and applied a forward genetic approach to decipher the signaling pathways activated by TLRs. He also identified many other molecules with non-redundant function in the immune response.

Beutler is currently a Regental Professor and Director of the Center for Genetics of Host Defense at the University of Texas Southwestern Medical Center. He also holds the Raymond and Ellen Willie Distinguished Chair in Cancer Research in honor of Laverne and Raymond Willie, Sr. He has authored or co-authored more than 300 papers, which have been cited more than 46,000 times. Before he received the Nobel Prize, his work was recognized by the Shaw Prize (2011), the Albany Medical Center Prize in Medicine and Biomedical Research (2009), election to the National Academy of Sciences and Institute of Medicine (2008), the Frederik B. Bang Award (2008), the Balzan Prize (2007), the Gran Prix Charles-Leopold-Mayer (2006), the William B. Coley Award (2005), the Robert-Koch-Prize (2004), and other honors.

“Monitored Saturation Mutagenesis of the Mouse Genome”

Bruce Beutler, University of Texas Southwestern Medical Center, TX USA

The availability of massively parallel sequencing platforms has accelerated the identification of induced mutations in the mouse genome. However, genetic mapping has remained essential to identify causative mutations, and for a time, became the rate-limiting step in mammalian forward genetics. We have recently described a procedure that permits real time identification of mutations responsible for phenotypes of any kind. By inducing mutations on a defined genetic background (C57BL/6J) and sequencing the whole exome of all G1 carriers of these mutations, we are able to identify all candidate aberrations that might cause phenotype in descendants of the G1. We then genotype all G3 mice to determine zygosity at each mutation site. Pre-genotyped G3 mice are distributed for extensive phenotypic screening. As quantitative phenotypic data are uploaded to the computer, an automated search for correlation between phenotype and genotype is initiated using dominant, recessive and semidominant models of transmission. The computer returns a list of statistical associations between specific mutations and phenotype, allowing immediate inference regarding causation. As allelic series develop at most loci, the strength of inference increases. CRISPR/Cas9 targeting is used to validate candidates identified by automated mapping. Presently, more than 65,000 point mutations disrupting the mouse coding region have been tested in this manner. To our best estimate, 18 percent of all mouse genes have been mutated to phenovariance and examined in 135 phenotypic screens. About 50,000 mutations can be surveyed annually. A list of robust associations between mouse genes and phenotypic anomalies will facilitate genetic investigations in humans, where a uniform genetic background is not available and a heavy mutation load confounds such analysis.

 

Nobel Laureate Bruce Beutler, Guest Lecturer at the First Annual …

http://www.youtube.com/watch%3Fv%3D31rWtdpmjME  
Dec 9, 2014 … Professor Bruce Beutler, a 2011 recipient of the Nobel Prize in Medicine, was the Keynote speaker. Professor Beutler discovered the …

 

In the last few years of his life, Dr. Ralph Steinman made himself into an extraordinary human lab experiment, testing a series of unproven therapies – including some he helped to create – as he waged a very personal battle with pancreatic cancer.

The winner of the 2011 Nobel prize in medicine died only three days before the award was announced.

Immunologist Ralph Steinman was honored today by the Nobel Committee for his discovery of dendritic cells, a class of immune cells that help rally the body’s natural defenses to fight disease. However, the prize is bittersweet for all who knew the 68-year-old scientist during his long career as a researcher and mentor at Rockefeller University in New York City.

“Dendritic cells are the guys who are training the fighters,” says Pawel Kalinski, an immunologist at the University of Pittsburgh in Pennsylvania, about their role in activating T cells, the body’s immune sentries. Over decades, with Steinman often leading the way, the work transformed cancer research. Cancer vaccines either using or targeting dendritic cells are now the subject of numerous clinical trials, and the first-ever cancer vaccine to be approved in the United States—called Provenge, to treat prostate cancer—injects a patients’ own dendritic cells back into their body. It went on the market last year.

“He felt that human clinical investigation was the highest form of research, that it was critical to engage in it,” Dr. Sarah Schlesinger, Steinman’s clinical lab director and colleague at New York’s Rockefeller University, told Reuters. “He had great criticism of how slowly the process moved … he was impatient with data and mice,” she added.

 

Steinman spent his entire career on immunology research for which he won the Nobel Prize, an honor he shares with American Bruce Beutler and French biologist Jules Hoffmann for their contributions to explaining the immune system.

Steinman’s discovery of dendritic cells in 1973 led to the first therapeutic cancer vaccine, Dendreon’s Provenge, which treats men with advanced prostate cancer.

 

Jules A. Hoffmann (born 2 August 1941) is a Luxembourg-born French^[1] biologist. During his youth, growing up in Luxembourg, he developed a strong interest in insects under the influence of his father, Jos Hoffmann. This eventually resulted in the younger Hoffmann’s dedication to the field of biology using insects as model organisms.^[2] He currently holds a faculty position at the University of Strasbourg.^[3] He is a research director and member of the board of administrators of the National Center of Scientific Research (CNRS) in Strasbourg, France. He was elected to the positions of Vice-President (2005-2006) and President (2007-2008) of the French Academy of Sciences.^[3] Hoffmann and Bruce Beutler were jointly awarded a half share of the 2011 Nobel Prize in Physiology or Medicine for “their discoveries concerning the activation of innate immunity,”.^[4] [More specifically,the work showing increased Drosomycin expression following activation of Toll pathway in microbial infection.]

Hoffmann and Lemaitre discovered the function of the fruit fly Toll gene in innate immunity. Its mammalian homologs, the Toll-like receptors, were discovered by Beutler. Toll-like receptors identify constituents of other organisms like fungi and bacteria, and trigger an immune response, explaining, for example, how septic shock can be triggered by bacterial remains.^[5][6][7]

 

During his Ph.D. program under Pierre Joly, Hoffmann started his research in studying antimicrobial defenses in grasshoppers, inspired by the previous works done in the laboratory of Pierre Joly showing that no opportunistic infections were apparent in insects after the transplantation of certain organs from one to another.^[2] Hoffmann confirmed discovery of phagocytosis done by Eli Metchnikoff, through injection of Bacillus thuringiensis and observation of increase of phagocytes.^[2] In addition, he showed strong correlation between hematopoiesis and antimicrobial defenses by assessing the susceptibility of an insect to the microbial infection after X-ray treatment.^[2] Hoffmann shifts from using grasshopper model to using dipteran species in the 80s. By using Phormia terranovae, Hoffmann and his colleagues were able to identify 82-residues long antimicrobial polypeptide named Diptericin which was glycine-rich, along with other polypeptides in Drosophila melanogaster such as defensin, cecropin, and attacin.^[2] Further molecular genetic analysis revealed that the promoters for the genes encoding these antimicrobial peptides contained DNA sequences similar to the binding elements for NF-κB in mammalian DNA. Dorsal gene, critical in dorso-ventral patterning in the early embryo of Drosophila melanogaster was also identified to be in this NF-κB family. It was initially speculated by Hoffmann and colleagues that activity of Dorsal was directly linked to the expression of the Diptericin gene. However, it turned out that Diptericin was normally induced even in the loss-of-function Dorsal mutants. Further conducted research showed that Diptericin expression was dependent on the expression of imd gene. Identification of another antifungal peptide named Drosomycin and RNA blots demonstrated that two distinct pathways(Toll, Imd) exist, involving Drosomycin and Diptericin respectively. Similarities of structure and function between several members in the Drosophila embryo and members in mammals being noted, study “The Dorsoventral Regulatory Gene Cassette spảtzle/Toll/cactus Controls the Potent Antifungal Response in Drosophila Adults”^[8] by Lemaitre and Hoffmann in 1996 illuminated the possible existing innate immunity in Drosophila in response to fungal challenge. Later works identified that Toll transmembrane receptors are present in a wide variety of phyla and are conserved through evolution along with conservation of NF-κB activating cascades.^[2]

Hoffmann was a research assistant at CNRS from 1964 to 1968, and became a research associate in 1969. Since 1974 he has been a Research Director of CNRS. Between 1978 and 2005 he was Director of the CNRS research unit “Immune Response and Development in Insects”, and from 1994 to 2005 he was director of the Institute of Molecular and Cellular Biology of CNRS in Strasbourg.

 

The History of Toll-like receptors — Redefining Innate Immunity

• Luke A. J. O’Neill, Douglas Golenbock & Andrew G. Bowie

Nature Reviews Immunology13,453–460(2013)  http://dx.doi.org:/10.1038/nri3446

The discovery of Toll-like receptors (TLRs) was an important event for immunology research and was recognized as such with the awarding of the 2011 Nobel Prize in Physiology or Medicine to Jules Hoffmann and Bruce Beutler, who, together with Ralph Steinman, the third winner of the 2011 Nobel Prize and the person who discovered the dendritic cell, were pioneers in the field of innate immunity. TLRs have a central role in immunity — in this Timeline article, we describe the landmark findings that gave rise to this important field of research.

Toll–like receptor signalling : Article : Nature Reviews Immunology

http://www.nature.com/articles/nri1391

Toll–like receptors in innate immunity – International Immunology

http://intimm.oxfordjournals.org/content/17/1/1

Toll–like Receptors and Their Crosstalk with Other Innate Receptors …

http://www.sciencedirect.com/science/article/pii/S1074761311001907

 

Toll-like Receptors and Their Crosstalk with Other Innate Receptors in Infection and Immunity

Taro Kawai & Shizuo Akira
Immunity  27 May 2011;   34(5): 637–650,  http://dx.doi.org/10.1016/j.immuni.2011.05.006
http://www.cell.com/immunity/abstract/S1074-7613(11)00190-7

oll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that play a central role in host cell recognition and responses to microbial pathogens. TLR-mediated recognition of components derived from a wide range of pathogens and their role in the subsequent initiation of innate immune responses is widely accepted; however, the recent discovery of non-TLR PRRs, such as C-type lectin receptors, NOD-like receptors, and RIG-I-like receptors, suggests that many aspects of innate immunity are more sophisticated and complex. In this review, we will focus on the role played by TLRs in mounting protective immune responses against infection and their crosstalk with other PRRs with respect to pathogen recognition.

Invivogen – Review

Toll-Like Receptors (TLRs) play a critical role in the early innate immune response to invading pathogens by sensing microorganism and are involved in sensing endogenous danger signals.

TLRs are evolutionarily conserved receptors are homologues of the Drosophila Toll protein, discovered to be important for defense against microbial infection [1]. TLRs recognize highly conserved structural motifs known as pathogen-associated microbial patterns (PAMPs), which are exclusively expressed by microbial pathogens,
or danger-associated molecular patterns (DAMPs) that are endogenous molecules released from necrotic or dying cells.

PAMPs include various bacterial cell wall components such as lipopolysaccharide (LPS),peptidoglycan (PGN) and lipopeptides, as well as flagellin, bacterial DNA and viral double-stranded RNA. DAMPs include intracellular proteins such as heat shock proteins as well as protein fragments from the extracellular matrix.

Stimulation of TLRs by the corresponding PAMPs or DAMPs initiates signaling cascades leading to the activation of transcription factors, such as AP-1, NF-κB and interferon regulatory factors (IRFs). Signaling by TLRs result in a variety of cellular responses including the production of interferons (IFNs), pro-inflammatory cytokines and effector cytokines that direct the adaptive immune response.

 

The TLR Family

TLRs are type I transmembrane proteins characterized by an extracellular domain containing leucine-rich repeats (LRRs) and a cytoplasmic tail that contains a conserved region called the Toll/IL-1 receptor (TIR) domain.  The structure of the extracellular domain of TLR3 was revealed by crystallography studies as a large horseshoe-shape [2].
TLRs are predominantly expressed in tissues involved in immune function, such as spleen and peripheral blood leukocytes, as well as those exposed to the external environment such as lung and the gastrointestinal tract.
Their expression profiles vary among tissues and cell types. TLRs are located on the plasma membrane with the exception of TLR3, TLR7, TLR9 which are localized in the endosomal compartment [3].

Ten human and twelve murine TLRs have been characterized, TLR1 to TLR10 in humans, and TLR1 toTLR9, TLR11, TLR12 and TLR13 in mice, the homolog of TLR10 being a pseudogene.

TLR2 is essential for the recognition of a variety of PAMPs from Gram-positive bacteria, including bacterial lipoproteins, lipomannans and lipoteichoic acids. TLR3 is implicated in virus-derived double-stranded RNA. TLR4 is predominantly activated by lipopolysaccharide. TLR5 detectsbacterial flagellin and TLR9 is required for response to unmethylated CpG DNA. Finally, TLR7 andTLR8 recognize small synthetic antiviral molecules [4], and single-stranded RNA was reported to be their natural ligand [5]. TLR11(12) has been reported to recognize uropathogenic E.coli [6] and a profilin-like protein from Toxoplasma gondii [7].

The repertoire of specificities of the TLRs is apparently extended by the ability of TLRs to heterodimerize with one another. For example, dimers of TLR2 and TLR6 are required for responses to diacylated lipoproteins while TLR2 and TLR1 interact to recognize triacylated lipoproteins [8]. Specificities of the TLRs are also influenced by various adapter and accessory molecules, such as MD-2 and CD14 that form a complex with TLR4 in response to LPS [9].

 

TLR Signaling

TLR signaling consists of at least two distinct pathways: a MyD88-dependent pathway that leads to the production of inflammatory cytokines, and a MyD88-independent pathway associated with the stimulation of IFN-β and the maturation of dendritic cells.

The MyD88-dependent pathway is common to all TLRs, except TLR3 [10]. Upon activation byPAMPs or DAMPs, TLRs hetero- or homodimerize inducing the recruitment of adaptor proteins via the cytoplasmic TIR domain.
Adaptor proteins include the TIR-domain containing proteins, MyD88, TIRAP (TIR-associated protein), Mal (MyD88 adaptor-like protein), TRIF (TIR domain-containing adaptor protein-inducing IFN-β) and TRAM (TRIF-related adaptor molecule).
Recruitment of MyD88 for instance, in turn recruits IRAK1 and IRAK4. IRAK4 subsequently activates IRAK1 by phosphorylation. Both IRAK1 and IRAK4 leave the MyD88-TLR complex and associate temporarily with TRAF6 leading to its ubiquitination. Bcl10 and MALT1 form oligomers that bind to TRAF6 promoting TRAF6 self-ubiquitination [11].
Recently, IRAK2 was shown to play a central role in TRAF6 ubiquitination [12]. Following ubiquitination, TRAF6 forms a complex with TAB2/TAB3/TAK1 inducing TAK1 activation [13]. TAK1 then couples to the IKK complex, which includes the scaffold protein NEMO, leading to the phosphorylation of IκB and the subsequent nuclear localization of NF-κB. Activation of NF-κB triggers the the production of pro-inflammatory cytokines such as TNF-α, IL-1 and IL-12.

Individual TLRs induce different signaling reponses by usage of the different adaptor molecules.
TLR4 and TLR2 signaling requires the adaptor TIRAP/Mal, which is involved in the MyD88-dependent pathway [14]. TLR3 triggers the production of IFN-β in response to double-stranded RNA, in a MyD88-independent manner, through the adaptor TRIF/TICAM-1 [15]. TRAM/TICAM-2 is another adaptor molecule involved in the MyD88-independent pathway [5] which function is restricted to the TLR4 pathway [16].

TLR3, TLR7, TLR8 and TLR9 recognize viral nucleic acids and induce type I IFNs. The signaling mechanisms leading to the induction of type I IFNs differ depending on the TLR activated. They involve the interferon regulatory
factors, IRFs, a family of transcription factors known to play a critical role in antiviral defense, cell growth and immune regulation. Three IRFs (IRF3, IRF5 and IRF7) function as direct transducers of virus-mediated TLR signaling. TLR3 and TLR4 activate IRF3 and IRF7 [17], while TLR7 and TLR8 activate IRF5 and IRF7 [18]. Furthermore, type I IFN production stimulated by TLR9 ligand CpG-A has been shown to be mediated by PI(3)K and mTOR [19].

 

TLR-Targeted Therapeutics

Significant progress has been made over the past years in the understanding of TLR function [20]. TLRs are essential receptors in host defense against pathogens by activating the innate immune system, a prerequisite to the induction of adaptive immune responses.

Although TLR-mediated signaling is paramount in eradicating microbial infections and promoting tissue repair, the regulation must be tight. TLRs are implicated in a number of inflammatory and immune disorders and play a role in cancer [21].
Many single nucleotide polymorphisms have been identified in various TLR genes and are associated with particular diseases. Several therapeutic agents targeting the TLRs are now under pre-clinical and clinical
evaluation [22].

However, the complexity lies in that TLRs act as double-edged swords either promoting or inhibiting disease progression. Furthermore, therapeutic agents targeting the TLRs must be able to antagonize the harmful effects resulting without affecting host defense functions.
Nonetheless, the potential of harnessing and directing the innate immune system with drugs targeting TLRs, to prevent or treat human inflammatory and autoimmune diseases as well as cancer, appears to be promising.

 

TLR	Immune Cell Expression	PAMPs	DAMPs	Signal Adaptor	Production
TLR1+ TLR2	Cell surface Mo, MΦ, DC, B	Triacylated lipoproteins (Pam3CSK4) Peptidoglycans,Lipopolysaccharides	(TLR2 DAMPs listed below)	TIRAP, MyD88, Mal	IC
TLR2+ TLR6	Cell surface Mo, MΦ, MC, B	Diacylated lipoproteins (FSL-1)	Heat Shock Proteins (HSP 60, 70, Gp96) High mobility group proteins (HMGB1) Proteoglycans (Versican, Hyaluronic Acid fragments)	TIRAP, MyD88, Mal	IC
TLR3	Endosomes B, T, NK, DC	dsRNA (poly (I:C)) tRNA, siRNA	mRNA tRNA	TRIF	IC, type1 IFN
TLR4	Cell surface/ endosomes Mo, MΦ, DC, MC, IE	Lipopolysaccharides (LPS) Paclitaxel	Heat Shock Proteins (HSP22, 60, 70,72, Gp96) High mobility group proteins (HMGB1) Proteoglycans (Versican, Heparin sulfate, Hyaluronic Acid fragments) Fibronectin, Tenascin-C	TRAM, TRIF TIRAP, MyD88 Mal	IC, type1 IFN
TLR5	Cell surface Mo, MΦ, DC, IE	Flagellin		MyD88	IC
TLR7	Endosomes Mo, MΦ, DC. B	ssRNA Imidazoquinolines (R848) Guanosine analogs (Loxoribine)	ssRNA	MyD88	IC, type1 IFN
TLR8	Endosomes Mo, MΦ, DC, MC	ssRNA, Imidazoquinolines (R848)	ssRNA	MyD88	IC, type1 IFN
TLR9	Endosomes Mo, MΦ, DC, B,T	CpG DNA CpG ODNs	Chromatin IgG complex	MyD88	IC, type1 IFN
TLR10	Endosomes Mo, MΦ, DC	profilin-like proteins		MyD88	IC

Mo: monocytes, MΦ: marcophages, DC: dendritic cells, MC: Mast cells, B: B cells, T: T cells, IE: Intestinal epithelium, IC: Inflammatory cytokines

 

 

1. Medzhitov R. et al., 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature, 388(6640):394-7.
2. Choe J. et al., 2005. Crystal structure of human Toll-like receptor 3 (TLR3) ectodomain. Science 309; 581-585.
3. Nishiya T. & DeFranco AL., 2004. Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling proper ties of the Toll-like receptors. J Biol Chem. 279(18):19008-17.
4. Jurk M. et al., 2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol, 3(6):499.
5. Heil F. et al., 2004. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science. 303(5663):1526-9.
6. Zhang D. et al., 2004. A toll-like receptor that prevents infection by uropathogenic bacteria. Science. 303:1522-1526.
7. Lauw FN. et al., 2005. Of mice and man: TLR11 (finally) finds profilin. Trends Immunol. 26(10):509-11.
8. Ozinsky A. et al., 2000.The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. PNAS USA, 97(25):13766-71.
9. Miyake K., 2003. Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles for MD-2. Int Immunopharmacol. 3(1):119-28.
10. Adachi O. et al., 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity. 9(1):143-50.
11. Sun L. et al., 2004. The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol Cell. 2004 14(3):289-301.
12. Keating SE. et al., 2007. IRAK-2 participates in multiple Toll-like receptor signaling pathways to NFκB via activation of TRAF6 ubiquitination. J Biol Chem. 282: 33435-33443.
13. Kanayama A. et al., 2004. TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains. Mol Cell. 15(4):535-48.
14. Horng T. et al., 2002.The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature. 420(6913):329- 33.
15. Yamamoto M. et al., 2002. Cutting edge: a novel Toll/IL-1 receptor domain-containing adaptor that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J Immunol. 169(12):6668-72.
16. Yamamoto M. et al., 2003. TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway. Nat Immunol. 4(11):1144-50.
17. Doyle S. et al., 2002. IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity. 17(3):251-63.
18. Schoenemeyer A. et al., 2005. The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling. J Biol Chem. 280(17):17005-12.
19. Costa-Mattioli M. & Sonenberg N. 2008. RAPping production of type I interferon in pDCs through mTOR. Nature Immunol. 9: 1097-1099.
20. Kawai T. & Akira S., 2011. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity Immunity 34(5):637-50.
21. Rakoff-Nahoum S. & Medzhitov R., 2009. Toll-like receptors and cancer. Nat Revs Cancer 9:57- 63.
22. Hennessy E. et al., 2010. Targeting Toll-like receptors: emerging therapeutics? Nat Rev Drug Discov 9(4) 293-307.

 

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CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Cell Biology, Signaling & Cell Circuits, Conference Coverage with Social Media, CRISPR/Cas9 & Gene Editing, Disease Biology, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Drug Delivery Platform Technology, Drug Toxicity, Gene Regulation, Gene Regulation and Evolution, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Metabolism, Metabolomics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Drug Discovery, Pharmacodynamics and Pharmacokinetics, Pharmacogenomics, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged anti-cancer drugs, Cancer - General, CRISPR-Cas9, Duodna, FDA, gene editing, gene expression, genetics, genome, Genome engineering, health, MIT, mouse model, research, science, tumor model on September 2, 2015| Leave a Comment »

CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development

  UPDATED 6/11/2021

CRISPR Diagnostics: CRISPR-dx Comes of Age: Tool in Drug Development

The past five years has seen a rapid expansion of the ability of CRISPR based tools toward diagnostic testing. Recently, CRISPR has been used to detect SARS-CoV-2 in patients. An article in the journal Science describes the different classes of CRISPR diagnostics in use today .

Update near end of post

UPDATED 8/08/2020

Association to Causation: Using GWAS to Identify Druggable Targets

A Gen Webinar Thursday, August 6, 2020; 11:00am – 12:30pm EST

See at end of post

Curator: Stephen J. Williams, Ph.D.

• 

 

CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA.

CRISPR/Cas systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids such as viruses by cleaving the foreign DNA in a sequence-dependent manner. Although CRISPR arrays were first identified in the Escherichia coli genome in 1987 (Ishino et al., 1987), their biological function was not understood until 2005, when it was shown that the spacers were homologous to viral and plasmid sequences suggesting a role in adaptive immunity (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005). Two years later, CRISPR arrays were confirmed to provide protection against invading viruses when combined with Cas genes (Barrangou et al., 2007). The mechanism of this immune system based on RNA-mediated DNA targeting was demonstrated shortly thereafter (Brouns et al., 2008; Deltcheva et al., 2011; Garneau et al., 2010; Marraffini and Sontheimer, 2008).

Jennifer Doudna, PhD Professor of Molecular and Cell Biology and Chemistry, University of California, Berkeley Investigator, Howard Hughes Medical Institute has recently received numerous awards and accolades for the discovery of CRISPR/Cas9 as a tool for mammalian genetic manipulation as well as her primary intended research target to understand bacterial resistance to viral infection.

A good post on the matter and Dr. Doudna can be seen below:

http://pharmaceuticalintelligence.com/2014/06/13/215-245-6132014-jennifer-doudna-the-biology-of-crisprs-from-genome-defense-to-genetic-engineering/

In Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting inheritable metabolic disorders in which may benefit from a CRISPR-Cas9 mediated therapy is discussed. However this curation is meant to focus on CRISPR/CAS9 AS A TOOL IN PRECLINICAL DRUG DEVELOPMENT.

 

Three Areas of Importance of CRISPR/Cas9 as a TOOL in Preclinical Drug Discovery Include:

1. Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE
2. CRISPR/CAS9 Use in Developing Models of Disease
3. CRISPR/CAS9 Use as a Diagnostic Tool

• Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES

I.     Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE

The advent of the first tools for manipulating genetic material (cloning, PCR, transgenic technology, and before microarray and other’omic methods) allowed scientists to probe novel, individual gene functions as well as their variants and mutants in a “one-gene-at-a time” process. In essence, a gene (or mutant gene) was sequenced, cloned into expression vectors and transfected into recipient cells where function was evaluated.

However, some of the experimental issues with this methodology involved

• Lack of knowledge of insertion site of the transgene – this leads to off-target effects usually due to insertion of a transgene in front of unwanted promoters or insertion at a site resulting in gene disruption or even mutagenesis. In an extreme case, such as transposon-induced mutagenesis may lead to transformation as described in an earlier post on this site How Mobile Elements in “Junk” DNA Promote Cancer – Part 1: Transposon-mediated Tumorigenesis

• Most transfections experiments result in NON ISOGENIC cell lines – by definition the insertion of a transgene alters the genetic makeup of a cell line. Simple transfection experiments with one transgene compared to a “null” transfectant compares non-isogenic lines, possibly confusing the interpretation of gene-function studies. Therefore a common technique is to develop cell lines with inducible gene expression, thereby allowing the investigator to compare a gene’s effect in ISOGENIC cell lines.

1. Use of CRSPR in Highthrough-put Screening of Genetic Function

A very nice presentation and summary of CRSPR’s use in determining gene function in a high-throughput manner can be found below

www.rna.uzh.ch/events/journalclub/20140429JCCaihong.pdf

1. Determining Off-target Effects of Gene Therapy Simplified with CRSPR

In GUIDE-seq: First genome-wide method of detecting off-target DNA breaks induced by CRISPR-Cas nucleases (from This Journal’s series on Live Meeting Coverage) at a 2014 Koch lecture

Shengdar Q Tsai and J Keith Joung describe

“

an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint ‘hotspots’.

SOURCE http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3117.html

“

II. CRISPR/Cas9 Use in Developing Models of Disease

 

1. Developing Animal Tumor Models

In a post this year I discussed a talk at the recent 2015 AACR National Meeting on a laboratories ability to use CRISPR gene editing in-vivo to produce a hepatocarcinoma using viral delivery. The post can be seen here: Notes from Opening Plenary Session – The Genome and Beyond from the 2015 AACR Meeting in Philadelphia PA; Sunday April 19, 2015

1) In this talk Dr. Tyler Jacks discussed his use of CRSPR to generate a mouse model of liver tumor in an immunocompetent mouse. Some notes from this talk are given below

1. B) Engineering Cancer Genomes: Tyler Jacks, Ph.D.; Director, Koch Institute for Integrative Cancer Research

• Cancer GEM’s (genetically engineered mouse models of cancer) had moved from transgenics to defined oncogenes
• Observation that p53 -/- mice develop spontaneous tumors (lymphomas)
• then GEMs moved to Cre/Lox systems to generate mice with deletions however these tumor models require lots of animals, much time to create, expensive to keep;
• figured can use CRSPR/Cas9 as rapid, inexpensive way to generate engineered mice and tumor models
• he used CRSPR/Cas9 vectors targeting PTEN to introduce PTEN mutations in-vivo to hepatocytes; when they also introduced p53 mutations produced hemangiosarcomas; took ONLY THREE months to produce detectable tumors
• also produced liver tumors by using CRSPR/Cas9 to introduce gain of function mutation in β-catenin

See an article describing this study by MIT News “A New Way To Model Cancer: New gene-editing technique allows scientists to more rapidly study the role of mutations in tumor development.”

The original research article can be found in the August 6, 2014 issue of Nature[1]

And see also on the Jacks Lab site under Research

2)     In the Upcoming Meeting New Frontiers in Gene Editing multiple uses of CRISPR technology is discussed in relation to gene knockout/function studies, tumor model development and

New Frontiers in Gene Editing

Session Spotlight:
BUILDING IN VIVO MODELS FOR DRUG DISCOVERY

Genome Editing Animal Models in Drug Discovery
Myung Shin, Ph.D., Senior Principal Scientist, Biology-Discovery, Genetics and Pharmacogenomics, Merck Research Laboratories

Recent advances in genome editing have greatly accelerated and expanded the ability to generate animal models. These tools allow generating mouse models in condensed timeline compared to that of conventional gene-targeting knock-out/knock-in strategies. Moreover, the genome editing methods have expanded the ability to generate animal models beyond mice. In this talk, we will discuss the application of ZFN and CRISPR to generate various animal models for drug discovery programs.

In vivo Cancer Modeling and Genetic Screening Using CRISPR/Cas9
Sidi Chen, Ph.D., Postdoctoral Fellow, Laboratories of Dr. Phillip A. Sharp and Dr. Feng Zhang, Koch Institute for Integrative Cancer Research at MIT and Broad Institute of Harvard and MIT

Here we describe a genome-wide CRISPR-Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library. The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late stage primary tumors were found to target a small set of genes, suggesting specific loss-of-function mutations drive tumor growth and metastasis.

FEATURED PRESENTATION: In vivo Chromosome Engineering Using CRISPR-Cas9
Andrea Ventura, M.D., Ph.D., Assistant Member, Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center

We will discuss our experience using somatic genome editing to engineer oncogenic chromosomal rearrangements in vivo. More specifically, we will present the results of our ongoing efforts aimed at modeling cancers driven by chromosomal rearrangements using viral mediated delivery of Crispr-Cas9 to adult animals.

RNAi and CRISPR/Cas9-Based in vivo Models for Drug Discovery
Christof Fellmann, Ph.D., Postdoctoral Fellow, Laboratory of Dr. Jennifer Doudna, Department of Molecular and Cell Biology, The University of California, Berkeley

Genetically engineered mouse models (GEMMs) are a powerful tool to study disease initiation, treatment response and relapse. By combining CRISPR/Cas9 and “Sensor” validated, tetracycline-regulated “miR-E” shRNA technology, we have developed a fast and scalable platform to generate RNAi GEMMs with reversible gene silencing capability. The synergy of CRISPR/Cas9 and RNAi enabled us to not only model disease pathogenesis, but also mimic drug therapy in mice, providing us capability to perform preclinical studies in vivo.

In vivo Genome Editing Using Staphylococcus aureus Cas9
Fei Ann Ran, Ph.D., Post-doctoral Fellow, Laboratory of Dr. Feng Zhang, Broad Institute and Junior Fellow, Harvard Society of Fellows

The RNA-guided Cas9 nuclease from the bacterial CRISPR/Cas system has been adapted as a powerful tool for facilitating targeted genome editing in eukaryotes. Recently, we have identified an additional small Cas9 nuclease from Staphylococcus aureus that can be packaged with its guide RNA into a single adeno-associated virus (AAV) vector for in vivo applications. We demonstrate the use of this system for effective gene modification in adult animals and further expand the Cas9 toolbox for in vivo genome editing.

OriGene, Making the Right Tools for CRISPR Research
Xuan Liu, Ph.D., Senior Director, Marketing, OriGene

CRISPR technology has quickly revolutionized the scientific community. Its simplicity has democratized the genome editing technology and enabled every lab to consider its utility in gene function research. As the largest tool box for gene functional research, OriGene created a large collection of CRISPR-related tools, including various all-in-one vectors for gRNA cloning, donor vector backbones, genome-wide knockout kits, AAVS1 insertion vectors, etc. OriGene’s high quality products will accelerate CRISPR research.

1. Transgenic Animals : Custom Mouse and Rat Model Generation Service Using CRISPR/Cas9 by AppliedStem Cell Inc. (http://www.appliedstemcell.com/)

A critical component of producing transgenic animals is the ability of each successive generations to pass on the transgene. In her post on this site, A NEW ERA OF GENETIC MANIPULATION  Dr. Demet Sag discusses the molecular biology of Cas9 systems and their efficiency to cause point mutations which can be passed on to subsequent generations

“

This group developed a new technology for editing genes that can be transferable change to the next generation by combining microbial immune defense mechanism, CRISPR/Cas9 that is the latest ground breaking technology for translational genomics with gene therapy-like approach.

• In short, this so-called “mutagenic chain reaction” (MCR) introduces a recessive mutation defined by CRISPR/Cas9 that lead into a high rate of transferable information to the next generation. They reported that when they crossed the female MCR offspring to wild type flies, the yellow phenotype observed more than 95 percent efficiency.

“

The advantage of CRISPR/Cas9 over ZFNs or TALENs is its scalability and multiplexibility in that multiple sites within the mammalian genome can be simultaneously modified, providing a robust, high-throughput approach for gene editing in mammalian cells.

Applied StemCell, Inc. offers various services related to animal models including conventional transgenic rats, and phenotype analysis using knock-in, knock-out strategies.

Further explanation of their use of CRSPR can be found at the site below:

http://pharmaceuticalintelligence.com/2014/10/29/gene-editing-at-crispr-speed-services-and-tools/

In addition, ReproCELL Inc., a Tokyo based stem cell company, uses CRSPR to develop

· Tailored disease model cells (hiPSC-Disease Model Cells)

• 2 types of services

• ReproUNUS™-g：human iPS cell derived functional cells involving gene editing by CRISPR/Cas9 system
• eproUNUS™-p：patient derived iPS cell derived functional cells

III. Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES

As of now it is unclear as to the strategy of pharma in how to use this technology for toxicology testing however a few companies have licensed the technology to use across their R&D platforms including

• AstraZeneca
• Thermo
• Novartis
• GE Healthcare

A recent paper used a sister technique TALEN to generate knock-in pigs which suggest that it would be possible to generate pigs with human transgenes, especially in human liver isozymes in orer to study hepatotoxicity of drugs.

Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs

Jing Yao, Jiaojiao Huang, Tang Hai, Xianlong Wang, Guosong Qin, Hongyong Zhang, Rong Wu, Chunwei Cao, Jianzhong Jeff Xi, Zengqiang Yuan, Jianguo Zhao

Sci Rep. 2014; 4: 6926. Published online 2014 November 5. doi: 10.1038/srep06926

UPDATED 8/08/2020

Association to Causation: Using GWAS to Identify Druggable Targets

A Gen Webinar Thursday, August 6⋅11:00am – 12:30pm

This webinar is available at https://www.genengnews.com/resources/webinars/association-to-causation-using-gwas-to-identify-druggable-target/

Speakers:

Martin Kampmann, PhD

Associate Professor
UCSF
Investigator
Chan Zuckerberg Biohub

Kevin Holden, PhD

Head of Science
Synthego

Abhi Saharia, PhD

VP, Commercial Development
Synthego

Human genetics provides perhaps the single best opportunity to innovate and improve clinical success rates, through the identification of novel drug targets for complex disease. Even as correlation identifies multiple genetic variants associated with disease, it is challenging to conduct requisite functional studies to identify the causal variants, especially since most association signals map to non-coding regions of the genome.

Genetic editing technologies, such as CRISPR, have enabled the modeling of associated variants at their native loci, including non-coding loci, empowering the identification of underlying biological mechanisms of disease with potential causal genes. However, genome editing is largely manual today severely limiting scale, and forcing the use of rational filters to prioritize which variants to investigate functionally.

In this GEN webinar, we will discuss several strategies enabling large-scale functional investigation of disease-associated variants in a cost- and time-effective manner, including different types of pooled CRISPR-based screens and the development of a fully automated genome engineering platform. We will also review how optimization of genome engineering on this platform enables the engineering of disease-associated variants at scale in pluripotent cells.

• They will be presenting on use of wide scale CRSPR screens to validate druggable targets
• The presenters will also discuss new platforms for these wide scale screens

Martin Kampmann, PhD UCSF

• Multiple genetic variants associated with disease
• Big gap between accumulation of genetic variant information and functions of these variants
• CRSPRi or CRSPa (siRNA coupled or enhancer coupled CRSPR guides)
• Arrayed screens: multiplate guide RNAs and phenotype measured (phenotype can be morphology, complex biological systems like organoids or non autonomous functions
• Using pooled screens and use of suitable cell model critical for this strategy
• For example in iPSC vs. neurons has different expression patterns upon same CRSPR of UBA1
• Advantage is using CRSPR to take iPSC from diseased variant patient to make a corrected isogenic control then introduce gRNAs and use modifier screens to determine phenotypes
• Generated a platform called CRISPRbrain.org to do bioinformatics on various experiments with different guide RNAs (CRSPRs)

Abhi Saharia, PhD Syntheco

• Target identification with CSRSPR at Scale
• Nature medicine paper did GWAS and found 27 SNV associated with high risk disease and a rational filter focused on 1 SNV in noncoding region but why study a single variant and if studied all 27 would they have been able to identify a more representative druggable set?
• Goal is to reduce or eliminate these rational filters
• HALO (scalable RNA guide), ECLIPSE platform (automated generation of modified cell lines, BIOINFORMATIC platform (integrated informatics)
• Syntheco uses an electroporation with ribonucleic proteins (RNP) to give highest efficiency and minimizes off target as complex is only in cells for a short period of time
• They confirm they are doing single cell cloning by using automated microscopy to confirm single cell growth in each cloning well

Kevin Holden, Head of Science at Syntheco

• Engineering iPSc genetically modified cells at scale
• The closer you get to your target site the more efficient your CRSPR so a big factor when making guides, especially for knock-in CRSPR
• Adding a small molecule non homologous end joining inhibitor increases efficiency to 95%
• Cold shocking the cells also assists in homologous repair
• Use cleavage resistant templates

III. CRISPR/CAS9 AS A DIAGNOSTIC TOOL

     In the journal Science, Omar Abudayyeh and Jonathan Gootenberg discuss how CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA).  In addition they discuss the work by Jiao et al. in combining this technique to develop a rapid and sensitive SARS-CoV2 diagnostic test.

Omar O. Abudayyeh, Jonathan S. Gootenberg. Science  28 May 2021: CRISPR Diagnostics
Vol. 372, Issue 6545, pp. 914-915; DOI: 10.1126/science.abi9335

Summary

Although clinical diagnostics take many forms, nucleic acid–based testing has become the gold standard for sensitive detection of many diseases, including pathogenic infections. Quantitative polymerase chain reaction (qPCR) has been widely adopted for its ability to detect only a few DNA or RNA molecules that can unambiguously specify a particular disease. However, the complexity of this technique restricts application to laboratory settings. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored the need for the development and deployment of nucleic acid tests that are economical, easily scaled, and capable of being run in low-resource settings, without sacrifices in speed, sensitivity or specificity. CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA). On page 941 of this issue, Jiao et al. (1) describe a new CRISPR-based tool to distinguish several SARS-CoV-2 variants in a single reaction.

Although clinical diagnostics take many forms, nucleic acid–based testing has become the gold standard for sensitive detection of many diseases, including pathogenic infections. Quantitative polymerase chain reaction (qPCR) has been widely adopted for its ability to detect only a few DNA or RNA molecules that can unambiguously specify a particular disease. However, the complexity of this technique restricts application to laboratory settings. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored the need for the development and deployment of nucleic acid tests that are economical, easily scaled, and capable of being run in low-resource settings, without sacrifices in speed, sensitivity or specificity. CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA). On page 941 of this issue, Jiao et al. (1) describe a new CRISPR-based tool to distinguish several SARS-CoV-2 variants in a single reaction.

There are multiple types of CRISPR systems comprising basic components of a single protein or protein complex, which cuts a specific DNA or RNA target programmed by a complementary guide sequence in a CRISPR-associated RNA (crRNA). The type V and VI systems and the CRISPR-associated endonucleases Cas12 (2, 3) and Cas13 (4, 5) bind and cut DNA or RNA, respectively. Furthermore, upon recognizing a target DNA or RNA sequence, Cas12 and Cas13 proteins exhibit “collateral activity” whereby any DNA or RNA, respectively, in the sample is cleaved regardless of its nucleic acid sequence (4, 6). Thus, reporter DNAs or RNAs, which allow for visual or fluorescent detection upon cleavage, can be added to a sample to infer the presence or absence of specific DNA or RNA species (4–8).

Initial versions of CRISPR-dx utilizing Cas13 alone were sensitive to the low picomolar range, corresponding to a limit of detection of millions of molecules in a microliter sample. To improve sensitivity, preamplification methods, such as recombinase polymerase amplification (RPA), PCR, loop-mediated isothermal amplification (LAMP), or nucleic acid sequence–based amplification (NASBA), can be used with Cas12 or Cas13 to enable a limit of detection down to a single molecule (8). This preamplification approach, applicable to both Cas12 and Cas13 (6, 7), enabled a suite of detection methods and multiplexing up to four orthogonal targets (7). Additional developments expanded CRISPR-dx readouts beyond fluorescence, including lateral flow (7), colorimetric (9), and electronic or material responsive readouts (10), allowing for instrument-free approaches. In addition, post–collateral-cleavage amplification methods, such as the use of the CRISPR-associated enzyme Csm6, have been combined with Cas13 to further increase the speed of CRISPR-dx tests (7). As an alternative to collateral-cleavage–based detection, type III CRISPR systems, which involve large multiprotein complexes capable of targeting both DNA and RNA, have been used for SARS-CoV-2 detection through production of colorimetric or fluorometric readouts (11).

FDA-authorized CRISPR-dx tests are currently only for use in centralized labs, because the most common CRISPR detection protocols require fluid handling steps and two different incubations, precluding their immediate use at the point of care. Single-step formulations have been developed to overcome this limitation, and these “one-pot” versions of CRISPR-dx are simple to run, operate at a single temperature, and run without complex equipment, producing either fluorescence or lateral flow readouts. The programmability of CRISPR makes new diagnostic tests easier to develop, and within months of the release of the SARS-CoV-2 genome, many COVID-19–specific CRISPR tests were reported and distributed around the world.

The broader capability for Cas enzyme–enhanced nucleic acid binding or cleavage has led to several other detection modalities. Cas9-based methods for cleaving nucleic acids in solution for diagnostic purposes have been combined with other detection platforms, such as destruction of undesired amplicons for preparation of next-generation sequencing libraries (12), or selective removal of alleles for nucleotide-specific detection (13). Alternatively, the programmable cleavage event from the Cas nuclease can be used to initiate an amplification reaction (14). Cas9-based DNA targeting has also been used for nucleotide detection in combination with solid-state electronics, promising an amplification-free platform for detection. In this platform, called CRISPR-Chip, the Cas9 protein binds nucleotide targets of interest (often in the context of the native genome) to graphene transistors, where the presence of these targets alters either current or voltage (15). By utilizing additional Cas9 orthologs and specific guide designs, CRISPR-Chip approaches have been tuned for single–base-pair sensitivity (15). Because they are integrated with electronic readers, CRISPR-Chip platforms may allow facile point-of-care detection with handheld devices.

 

Other related articles on CRISPR/Cas9 were published in this Open Access Online Scientific Journal, include the following:

Search Results for ‘CRISPR’

Where is the most promising avenue to success in Pharmaceuticals with CRISPR-Cas9?

CRISPR/Cas9 genome editing tool for Staphylococcus aureus Cas9 complex (SaCas9) @ MIT’s Broad Institute

Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting

Using CRISPR to investigate pancreatic cancer

Simple technology makes CRISPR gene editing cheaper

RNAi, CRISPR, and Gene Editing: Discussions on How To’s and Best Practices @14th Annual World Preclinical Congress June 10-12, 2015 | Westin Boston Waterfront | Boston, MA

CRISPR/Cas9: Contributions on Endoribonuclease Structure and Function, Role in Immunity and Applications in Genome Engineering

CRISPR-CAS editing brings cloning of woolly mammoth one step closer to reality

GUIDE-seq: First genome-wide method of detecting off-target DNA breaks induced by CRISPR-Cas nucleases

The Patents for CRISPR, the DNA editing technology as the Biggest Biotech Discovery of the Century

CRISPR: Applications for Autoimmune Diseases @UCSF

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Posted in Academic Publishing, Amino acids, Anaerobic Glycolysis, Biochemical pathways, Biological Networks, Biomarkers & Medical Diagnostics, Bone Disease and Musculoskeletal Disease, Ca2+ triggered activation, Cachexia, Calcium, Calcium Signaling, Calmodulin, Calmodulin Kinase and Contraction, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Cardiac & Vascular Repair Tools Subsegment, Cardiac and Cardiovascular Surgical Procedures, Cardiac muscle regeneration, Cardiomyopathy, Cardiovascular Pharmacogenomics, Cardiovascular Research, Cell Biology, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Clinical Diagnostics, Clinical Genomics, Competition in regenerative stem cell science, Curation, Curation methodology, Cytoskeleton, Dehydrogenase, Diabetes Mellitus, Discovery process, Disease Biology, Endocrine Diseases, Enzyme Induction, Enzymes and isoenzymes, Explanatory, Fatty acids, Folate and B12, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Gene Regulation and Evolution, Genome Biology, Genomic Endocrinology, Genomic Expression, Glycobiology: Biopharmaceutical Production, Health Economics and Outcomes Research, Hexokinase, Historical relevance, Inosine nucleotides, Interviews with Scientific Leaders, Iron deficiency, Kinase, Lipid metabolism, Lipids, Liver & Digestive Diseases Research, Loss of function gene, Metabolism, Metabolomics, Methylation, Mg++, Molecular Genetics & Pharmaceutical, mtDNA, Mutant Gene Expression, Myelodysplasia, Myelofibrosis, Myocardial Ischemia, Myocardial Metabolism, Na-K-ATPase, Neurodegenerative Diseases, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Nutrigenomics, Nutrition, Nutrition and Phytochemistry, Nutrition Disorders, Nutritional Supplements: Atherogenesis, Open Access Journals, Oxidative phosphorylation, Pentose monophosphate shunt, Phosphatase, Phosphorylase, Phosphorylation, PKC, Plant extract, Proteins, Proteomics, Pyridine nucleotide transhydrogenase, Pyridine nucleotides, Pyruvate Kinase, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, RNA Biology, S-nitrosylation, Signaling, Signaling & Cell Circuits, Skeletal muscle regeneration, Small Molecules in Development of Therapeutic Drugs, Theoretic convergence, Transcriptomics, Translational Science, Ubiquitinylation, Warburg effect, tagged ATP, Aviva Lev-Ari, Biology, Cell Biology, cell metabolic regulation, energy metabolism, gene, genetics, health, Heart disease, Larry H. Bernstein, Lipid metabolism, metabolic analysis, metabolic disorders, Metabolomics, Proteomics, research, science on August 15, 2015| Leave a Comment »

Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD

Article ID #180: Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle. Published on 8/15/2015

WordCloud Image Produced by Adam Tubman

Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

which is now available on Amazon Kindle at

http://www.amazon.com/dp/B012BB0ZF0.

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

http://pharmaceuticalintelligence.com/biomed-e-books/

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

• delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
• providing a social platform for scientists and clinicians to enter into discussion using social media
• compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 

 

Summary 

Epilogue

 

 

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Treatment for Chronic Leukemias [2.4.4B]

Posted in Academic Publishing, Automated Cell Processing, BioIT: BioInformatics, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Cancer Screening, Clinical & Translational, Clinical Genomics, Curation, Diagnostics and Lab Tests, Discovery process, Disease Biology, Experimental validation, Explanatory, Gene Regulation, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genomic Expression, Hematopoiesis, Historical relevance, Human Immune System in Health and in Disease, Molecular Genetics & Pharmaceutical, Mutant Gene Expression, Myelodysplasia, Myelofibrosis, Neutrophilia, Pharmaceutical Analytics, Pharmaceutical Drug Discovery, Platelet count disorder, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Technology Advance Assessment of, tagged Chemotherapy, chronic lymphocytic leukemia, Chronic myelogenous leukemia, myelodyspalstic syndromes on August 11, 2015| Leave a Comment »

Treatment for Chronic Leukemias [2.4.4B]

Larry H. Bernstein, MD, FCAP, Author, Curator, Editor

http://pharmaceuticalintelligence.com/2015/8/11/larryhbern/Treatment-for-Chronic-Leukemias-[2.4.4B]

2.4.4B1 Treatment for CML

Chronic Myelogenous Leukemia Treatment (PDQ®)

http://www.cancer.gov/cancertopics/pdq/treatment/CML/Patient/page4

Treatment Option Overview

Key Points for This Section

There are different types of treatment for patients with chronic myelogenous leukemia.

Six types of standard treatment are used:

1. Targeted therapy
2. Chemotherapy
3. Biologic therapy
4. High-dose chemotherapy with stem cell transplant
5. Donor lymphocyte infusion (DLI)
6. Surgery

New types of treatment are being tested in clinical trials.

Patients may want to think about taking part in a clinical trial.

Patients can enter clinical trials before, during, or after starting their cancer treatment.

Follow-up tests may be needed.

There are different types of treatment for patients with chronic myelogenous leukemia.

Different types of treatment are available for patients with chronic myelogenous leukemia (CML). Some treatments are standard (the currently used treatment), and some are being tested in clinical trials. A treatment clinical trial is a research study meant to help improve current treatments or obtain information about new treatments for patients with cancer. When clinical trials show that a new treatment is better than the standard treatment, the new treatment may become the standard treatment. Patients may want to think about taking part in a clinical trial. Some clinical trials are open only to patients who have not started treatment.

Six types of standard treatment are used:

Targeted therapy

Targeted therapy is a type of treatment that uses drugs or other substances to identify and attack specific cancer cells without harming normal cells. Tyrosine kinase inhibitors are targeted therapy drugs used to treat chronic myelogenous leukemia.

Imatinib mesylate, nilotinib, dasatinib, and ponatinib are tyrosine kinase inhibitors that are used to treat CML.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Chemotherapy

Chemotherapy is a cancer treatment that uses drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. When chemotherapy is taken by mouth or injected into a vein or muscle, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy). When chemotherapy is placed directly into the cerebrospinal fluid, an organ, or a body cavity such as the abdomen, the drugs mainly affect cancer cells in those areas (regional chemotherapy). The way the chemotherapy is given depends on the type and stage of the cancer being treated.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Biologic therapy

Biologic therapy is a treatment that uses the patient’s immune system to fight cancer. Substances made by the body or made in a laboratory are used to boost, direct, or restore the body’s natural defenses against cancer. This type of cancer treatment is also called biotherapy or immunotherapy.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

High-dose chemotherapy with stem cell transplant

High-dose chemotherapy with stem cell transplant is a method of giving high doses of chemotherapy and replacing blood-forming cells destroyed by the cancer treatment. Stem cells (immature blood cells) are removed from the blood or bone marrow of the patient or a donor and are frozen and stored. After the chemotherapy is completed, the stored stem cells are thawed and given back to the patient through an infusion. These reinfused stem cells grow into (and restore) the body’s blood cells.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Donor lymphocyte infusion (DLI)

Donor lymphocyte infusion (DLI) is a cancer treatment that may be used after stem cell transplant.Lymphocytes (a type of white blood cell) from the stem cell transplant donor are removed from the donor’s blood and may be frozen for storage. The donor’s lymphocytes are thawed if they were frozen and then given to the patient through one or more infusions. The lymphocytes see the patient’s cancer cells as not belonging to the body and attack them.

Surgery

Splenectomy

What`s new in chronic myeloid leukemia research and treatment?

http://www.cancer.org/cancer/leukemia-chronicmyeloidcml/detailedguide/leukemia-chronic-myeloid-myelogenous-new-research

Combining the targeted drugs with other treatments

Imatinib and other drugs that target the BCR-ABL protein have proven to be very effective, but by themselves these drugs don’t help everyone. Studies are now in progress to see if combining these drugs with other treatments, such as chemotherapy, interferon, or cancer vaccines (see below) might be better than either one alone. One study showed that giving interferon with imatinib worked better than giving imatinib alone. The 2 drugs together had more side effects, though. It is also not clear if this combination is better than treatment with other tyrosine kinase inhibitors (TKIs), such as dasatinib and nilotinib. A study going on now is looking at combing interferon with nilotinib.

Other studies are looking at combining other drugs, such as cyclosporine or hydroxychloroquine, with a TKI.

New drugs for CML

Because researchers now know the main cause of CML (the BCR-ABL gene and its protein), they have been able to develop many new drugs that might work against it.

In some cases, CML cells develop a change in the BCR-ABL oncogene known as a T315I mutation, which makes them resistant to many of the current targeted therapies (imatinib, dasatinib, and nilotinib). Ponatinib is the only TKI that can work against T315I mutant cells. More drugs aimed at this mutation are now being tested.

Other drugs called farnesyl transferase inhibitors, such as lonafarnib and tipifarnib, seem to have some activity against CML and patients may respond when these drugs are combined with imatinib. These drugs are being studied further.

Other drugs being studied in CML include the histone deacetylase inhibitor panobinostat and the proteasome inhibitor bortezomib (Velcade).

Several vaccines are now being studied for use against CML.

2.4.4.B2 Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia Treatment (PDQ®)

General Information About Chronic Lymphocytic Leukemia

Key Points for This Section

1. Chronic lymphocytic leukemia is a type of cancer in which the bone marrow makes too many lymphocytes (a type of white blood cell).
2. Leukemia may affect red blood cells, white blood cells, and platelets.
3. Older age can affect the risk of developing chronic lymphocytic leukemia.
4. Signs and symptoms of chronic lymphocytic leukemia include swollen lymph nodes and tiredness.
5. Tests that examine the blood, bone marrow, and lymph nodes are used to detect (find) and diagnose chronic lymphocytic leukemia.
6. Certain factors affect treatment options and prognosis (chance of recovery).
7. Chronic lymphocytic leukemia is a type of cancer in which the bone marrow makes too many lymphocytes (a type of white blood cell).

Chronic lymphocytic leukemia (also called CLL) is a blood and bone marrow disease that usually gets worse slowly. CLL is one of the most common types of leukemia in adults. It often occurs during or after middle age; it rarely occurs in children.

http://www.cancer.gov/images/cdr/live/CDR755927-750.jpg

Anatomy of the bone; drawing shows spongy bone, red marrow, and yellow marrow. A cross section of the bone shows compact bone and blood vessels in the bone marrow. Also shown are red blood cells, white blood cells, platelets, and a blood stem cell.

Anatomy of the bone. The bone is made up of compact bone, spongy bone, and bone marrow. Compact bone makes up the outer layer of the bone. Spongy bone is found mostly at the ends of bones and contains red marrow. Bone marrow is found in the center of most bones and has many blood vessels. There are two types of bone marrow: red and yellow. Red marrow contains blood stem cells that can become red blood cells, white blood cells, or platelets. Yellow marrow is made mostly of fat.

Leukemia may affect red blood cells, white blood cells, and platelets.

Normally, the body makes blood stem cells (immature cells) that become mature blood cells over time. A blood stem cell may become a myeloid stem cell or a lymphoid stem cell.

A myeloid stem cell becomes one of three types of mature blood cells:

1. Red blood cells that carry oxygen and other substances to all tissues of the body.
2. White blood cells that fight infection and disease.
3. Platelets that form blood clots to stop bleeding.

A lymphoid stem cell becomes a lymphoblast cell and then one of three types of lymphocytes (white blood cells):

1. B lymphocytes that make antibodies to help fight infection.
2. T lymphocytes that help B lymphocytes make antibodies to fight infection.
3. Natural killer cells that attack cancer cells and viruses.

Blood cell development. CDR526538-750

http://www.cancer.gov/images/cdr/live/CDR526538-750.jpg

Blood cell development; drawing shows the steps a blood stem cell goes through to become a red blood cell, platelet, or white blood cell. A myeloid stem cell becomes a red blood cell, a platelet, or a myeloblast, which then becomes a granulocyte (the types of granulocytes are eosinophils, basophils, and neutrophils). A lymphoid stem cell becomes a lymphoblast and then becomes a B-lymphocyte, T-lymphocyte, or natural killer cell.

Blood cell development. A blood stem cell goes through several steps to become a red blood cell, platelet, or white blood cell.

In CLL, too many blood stem cells become abnormal lymphocytes and do not become healthy white blood cells. The abnormal lymphocytes may also be called leukemia cells. The lymphocytes are not able to fight infection very well. Also, as the number of lymphocytes increases in the blood and bone marrow, there is less room for healthy white blood cells, red blood cells, and platelets. This may cause infection, anemia, and easy bleeding.

This summary is about chronic lymphocytic leukemia. See the following PDQ summaries for more information about leukemia:

• Adult Acute Lymphoblastic Leukemia Treatment.
• Childhood Acute Lymphoblastic Leukemia Treatment.
• Adult Acute Myeloid Leukemia Treatment.
• Childhood Acute Myeloid Leukemia/Other Myeloid Malignancies Treatment.
• Chronic Myelogenous Leukemia Treatment.
• Hairy Cell Leukemia Treatment

Older age can affect the risk of developing chronic lymphocytic leukemia.

Anything that increases your risk of getting a disease is called a risk factor. Having a risk factor does not mean that you will get cancer; not having risk factors doesn’t mean that you will not get cancer. Talk with your doctor if you think you may be at risk. Risk factors for CLL include the following:

• Being middle-aged or older, male, or white.
• A family history of CLL or cancer of the lymph system.
• Having relatives who are Russian Jews or Eastern European Jews.

Signs and symptoms of chronic lymphocytic leukemia include swollen lymph nodes and tiredness.

Usually CLL does not cause any signs or symptoms and is found during a routine blood test. Signs and symptoms may be caused by CLL or by other conditions. Check with your doctor if you have any of the following:

• Painless swelling of the lymph nodes in the neck, underarm, stomach, or groin.
• Feeling very tired.
• Pain or fullness below the ribs.
• Fever and infection.
• Weight loss for no known reason.

Tests that examine the blood, bone marrow, and lymph nodes are used to detect (find) and diagnose chronic lymphocytic leukemia.

The following tests and procedures may be used:

Physical exam and history : An exam of the body to check general signs of health, including checking for signs of disease, such as lumps or anything else that seems unusual. A history of the patient’s health habits and past illnesses and treatments will also be taken.

Complete blood count (CBC) with differential : A procedure in which a sample of blood is drawn and checked for the following:

The number of red blood cells and platelets.

The number and type of white blood cells.

The amount of hemoglobin (the protein that carries oxygen) in the red blood cells.

The portion of the blood sample made up of red blood cells.

Results from the Phase 3 Resonate™ Trial

Significantly improved progression free survival (PFS) vs ofatumumab in patients with previously treated CLL

• Patients taking IMBRUVICA® had a 78% statistically significant reduction in the risk of disease progression or death compared with patients who received ofatumumab¹
• In patients with previously treated del 17p CLL, median PFS was not yet reached with IMBRUVICA® vs 5.8 months with ofatumumab (HR 0.25; 95% CI: 0.14, 0.45)¹

Significantly prolonged overall survival (OS) with IMBRUVICA® vs ofatumumab in patients with previously treated CLL

• In patients with previously treated CLL, those taking IMBRUVICA® had a 57% statistically significant reduction in the risk of death compared with those who received ofatumumab (HR 0.43; 95% CI: 0.24, 0.79; P<0.05)¹

Typical treatment of chronic lymphocytic leukemia

http://www.cancer.org/cancer/leukemia-chroniclymphocyticcll/detailedguide/leukemia-chronic-lymphocytic-treating-treatment-by-risk-group

Treatment options for chronic lymphocytic leukemia (CLL) vary greatly, depending on the person’s age, the disease risk group, and the reason for treating (for example, which symptoms it is causing). Many people live a long time with CLL, but in general it is very difficult to cure, and early treatment hasn’t been shown to help people live longer. Because of this and because treatment can cause side effects, doctors often advise waiting until the disease is progressing or bothersome symptoms appear, before starting treatment.

If treatment is needed, factors that should be taken into account include the patient’s age, general health, and prognostic factors such as the presence of chromosome 17 or chromosome 11 deletions or high levels of ZAP-70 and CD38.

Initial treatment

Patients who might not be able to tolerate the side effects of strong chemotherapy (chemo), are often treated with chlorambucil alone or with a monoclonal antibody targeting CD20 like rituximab (Rituxan) or obinutuzumab (Gazyva). Other options include rituximab alone or a corticosteroid like prednisione.

In stronger and healthier patients, there are many options for treatment. Commonly used treatments include:

• FCR: fludarabine (Fludara), cyclophosphamide (Cytoxan), and rituximab
• Bendamustine (sometimes with rituximab)
• FR: fludarabine and rituximab
• CVP: cyclophosphamide, vincristine, and prednisone (sometimes with rituximab)
• CHOP: cyclophosphamide, doxorubicin, vincristine (Oncovin), and prednisone
• Chlorambucil combined with prednisone, rituximab, obinutuzumab, or ofatumumab
• PCR: pentostatin (Nipent), cyclophosphamide, and rituximab
• Alemtuzumab (Campath)
• Fludarabine (alone)

Other drugs or combinations of drugs may also be also used.

If the only problem is an enlarged spleen or swollen lymph nodes in one region of the body, localized treatment with low-dose radiation therapy may be used. Splenectomy (surgery to remove the spleen) is another option if the enlarged spleen is causing symptoms.

Sometimes very high numbers of leukemia cells in the blood cause problems with normal circulation. This is calledleukostasis. Chemo may not lower the number of cells until a few days after the first dose, so before the chemo is given, some of the cells may be removed from the blood with a procedure called leukapheresis. This treatment lowers blood counts right away. The effect lasts only for a short time, but it may help until the chemo has a chance to work. Leukapheresis is also sometimes used before chemo if there are very high numbers of leukemia cells (even when they aren’t causing problems) to prevent tumor lysis syndrome (this was discussed in the chemotherapy section).

Some people who have very high-risk disease (based on prognostic factors) may be referred for possible stem cell transplant (SCT) early in treatment.

Second-line treatment of CLL

If the initial treatment is no longer working or the disease comes back, another type of treatment may help. If the initial response to the treatment lasted a long time (usually at least a few years), the same treatment can often be used again. If the initial response wasn’t long-lasting, using the same treatment again isn’t as likely to be helpful. The options will depend on what the first-line treatment was and how well it worked, as well as the person’s health.

Many of the drugs and combinations listed above may be options as second-line treatments. For many people who have already had fludarabine, alemtuzumab seems to be helpful as second-line treatment, but it carries an increased risk of infections. Other purine analog drugs, such as pentostatin or cladribine (2-CdA), may also be tried. Newer drugs such as ofatumumab, ibrutinib (Imbruvica), and idelalisib (Zydelig) may be other options.

If the leukemia responds, stem cell transplant may be an option for some patients.

Some people may have a good response to first-line treatment (such as fludarabine) but may still have some evidence of a small number of leukemia cells in the blood, bone marrow, or lymph nodes. This is known as minimal residual disease. CLL can’t be cured, so doctors aren’t sure if further treatment right away will be helpful. Some small studies have shown that alemtuzumab can sometimes help get rid of these remaining cells, but it’s not yet clear if this improves survival.

Treating complications of CLL

One of the most serious complications of CLL is a change (transformation) of the leukemia to a high-grade or aggressive type of non-Hodgkin lymphoma called diffuse large cell lymphoma. This happens in about 5% of CLL cases, and is known as Richter syndrome. Treatment is often the same as it would be for lymphoma (see our document called Non-Hodgkin Lymphoma for more information), and may include stem cell transplant, as these cases are often hard to treat.

Less often, CLL may transform to prolymphocytic leukemia. As with Richter syndrome, these cases can be hard to treat. Some studies have suggested that certain drugs such as cladribine (2-CdA) and alemtuzumab may be helpful.

In rare cases, patients with CLL may have their leukemia transform into acute lymphocytic leukemia (ALL). If this happens, treatment is likely to be similar to that used for patients with ALL (see our document called Leukemia: Acute Lymphocytic).

Acute myeloid leukemia (AML) is another rare complication in patients who have been treated for CLL. Drugs such as chlorambucil and cyclophosphamide can damage the DNA of blood-forming cells. These damaged cells may go on to become cancerous, leading to AML, which is very aggressive and often hard to treat (see our document calledLeukemia: Acute Myeloid).

CLL can cause problems with low blood counts and infections. Treatment of these problems were discussed in the section “Supportive care in chronic lymphocytic leukemia.”

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