Article - Fixing Crispr

http://www.genengnews.com/gen-exclusives/fixing-crispr/77900928

• Elegant in its simplicity and easily accessible to most laboratories, CRISPR/Cas9 technology has been widely adopted as a DNA-editing tool in research labs, and a group of investigators in China has begun to use it in a clinical trial to disable the PD-1 gene in lung cancer patients to slow or stop the cancer’s progression. But the technology’s off-target effects have raised serious concerns about its ultimate applicability to human therapies.
  CRISPR proponents say it’s less clunky to use that other recently emerged genome-editing technologies, including zinc-finger nucleases (ZFNs) and transcription activator–like effector nucleases (TALENs). ZFNs and TALENs work by using a strategy of tethering endo­nuclease catalytic domains to modular DNA-binding proteins for inducing targeted DNA double-stranded breaks (DSBs) at specific genomic loci.
  In contrast, Cas9 is a nuclease guided by small RNAs through Watson-Crick base pairing with target DNA, described by F.A. Ran et al. as a system that is “markedly easier to design, highly specific, efficient, and well-suited for high throughput and multiplexed gene editing for a variety of cell types and organisms.”
  CRISPR/Cas9 nucleases consist of the Cas9 bacterial enzyme and a short, 20-nucleotide RNA molecule that matches the desired target DNA sequence. In addition to the RNA/DNA match, the Cas9 enzyme must recognize a specific nucleotide sequence called a protospacer adjacent motif (PAM) adjacent to the target DNA.
  The most commonly used form of Cas9, derived from the bacteria Streptococcus pyogenes and known as SpCas9, recognizes PAM sequences in which any nucleotide is followed by two guanine DNA bases. This limits the DNA sequences that can be targeted using SpCas9 only to those that include two sequential guanines. 

• Off Target Effects

  But almost as soon as the technology was introduced, scientists raised concerns about off target effects. Said Xiao-Hui Zhang et al. of the College of Veterinary Medicine, South China Agricultural University and coauthors at MIT in a 2015 Molecular Therapy—Nucleic Acids article, “The high frequency of off-target activity (≥50%)—RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site is one major concern, especially for CRISPR technology therapeutic and clinical applications.”
  The growth of any new technology, the authors note, including CRISPR/Cas9, demands progressive enhancement. And while research in CRISPR/Cas9 has made “huge strides in the evolution of gene editing,” it and other RGENs, which include ZFNs and TALENs, have more severe off-target effects than other nucleases due to their inherent structure and mechanism.
  At an American Society of Hematology workshop on genome editing, CRISPR pioneer J. Keith Joung, M.D., Ph.D., of Massachusetts General Hospital said, “In the early days of this field, algorithms were generated to predict off-target effects and [made] available on the web miss a fair number of off-target effects. He added, “These tools are used in a lot of papers, but they really aren’t very good at predicting where there will be off-target effects,” according to STAT.
  Observations in the recent literature have raised more alarms among CRISPR cognoscenti, all of whom would agree than technical improvements are needed. In one of the most blogged-about papers on CRISPR, “Unexpected mutations after CRISPR–Cas9 editing in vivo,” Kellie A. Schaefer and colleagues at Stanford concluded that “More work may be needed to increase the fidelity of CRISPR/Cas9 with regard to off-target mutation generation before the CRISPR platform can be used without risk, especially in the clinical setting.”
  The authors had, they reported in a 2016 study by W.H. Wu et al. in Molecular Therapy, used CRISPR/Cas9 for sight restoration in blind rd1 mice by correcting a mutation in the Pde6b gene. Mice homozygous for the rd mutation have hereditary retinal degeneration and have been considered a model for human retinitis pigmentosa.
  Citing persistent concerns about secondary mutations in regions not targeted by a single guide RNA (sgRNA)—concerns also expressed by a number of other scientists—Kellie A. Schaefer, Ph.D., at Stanford University and colleagues at Howard Hughes and Massachusetts General Hospital performed whole genome sequencing (WGS) on DNA isolated from two CRISPR-repaired mice (F03 and F05) and one uncorrected control.
  CRISPR/Cas9-treated mice were sequenced at an average depth of 50×, and the control to 30× to identify all off target mutations. The sequencing the authors said identified an “unexpectedly high” number of single nucleotide variations (SNVs), contrary to the widely accepted assumption that CRISPR causes mutations mostly at regions homologous to the sgRNA.
  CRISPR’s penchant for promiscuous behavior has spawned an entirely new research field focused on fixing it. Patents have already been filed on the fixes and the developers believe that these advances will incrementally enable more reliable CRISPER performance. Most efforts have concentrated on modifying CRISPR nuclease Cas9 using structure-design based changes in the enzyme, chemical modifications, and amino acid substitutions at critical sights to better predict and control its function.
  Writing in Nature in 2015, Benjamin P. Kleinstiver, Ph.D., of the Molecular Pathology Unit and Center for Cancer Research at the Massachusetts General Hospital and colleagues noted that although CRISPR/Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific PAM.
  The investigators reported that they could modify Cas9 to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. The altered PAM specificity variants, they said, could edit endogenous gene sites in zebrafish and human cells that are not targetable by wild-type SpCas9. Further, they said, the variants’ genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis and establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.
  Kleinstiver and other investigators working in Joung’s also developed the unique endonuclease SpCas9-HF1, which they describe as a high-fidelity enzyme variant with alterations designed to reduce non-specific DNA contacts. The scientists hypothesized that reducing iCas9 and the target DNA interactions might help eliminate off-target effects while still retaining the desired on-target interaction.
  Since certain portions of the Cas9 nuclease can itself interact with the backbone of the target DNA molecule, the team altered four of these Cas9-mediated contacts by replacing the long amino acid side-chains that bind to the DNA backbone with shorter ones that could not bind.
  SpCas9-HF1 retained on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard nonrepetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods.
  Jiang and Doudna pointed out in a 2017 piece in Annual Review of Biophysics how Cas9 locates specific 20-base-pair (bp) target sequences within the genomes that are millions to billions of base pairs long and, subsequently, how it induces sequence-specific double-stranded DNA (dsDNA) cleavage remain critical questions, not just in CRISPR biology, but in the efforts to develop more precise and efficient Cas9-based tools.
  Molecular insights from biochemical and structural studies such as those describe above will provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity and PAM requirements, and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

Hemophilia B Article - Genetic Literacy Project

https://geneticliteracyproject.org/2017/06/22/blood-disease-plagued-europes-royal-families-might-treatable-using-gene-editing/
It was once known as the disease that plagued royal families in the 19^th and 20^th centuries. Hemophilia B famously struck dynasties in England, Spain, Germany, and Russia, beginning with diminutive Queen Victoria, who ruled England from 1837 to 1901. The Queen’s life was depicted in the first season of a recent Masterpiece Theater series.

Queen Victoria’s parents had no family history of hemophilia, so she was likely a carrier due to a spontaneous mutation. She passed her mutation onto her son, Prince Leopold (a hemophiliac who died from his mutation at age 30 after a minor fall, but not before passing it along to his only daughter, Princess Alice of Albany), and to two of her daughters, Princess Alice and Princess Beatrice, who were carriers, who carried the mutation he mutation into the Spanish, German and Russian royal families.

Victoria’s granddaughter Alix married Tsar Nicholas, resulting in son Alexei. The child was executed at age 13 along with the rest of his family, but in 2009 his charred bones yielded DNA that revealed him to have the bleeding disorder.
The last living relative of the lineage was Prince Waldemar of Prussia, a cousin of the ill-fated Alexei, who died as World War II was ending and blood supplies were sent to the emptying concentration camps. He was denied a transfusion.

Today, researchers are looking to genome editing to provide a more robust treatment option for the disease sufferers. And for at least one company, those efforts involve harnessing the power of albumin – the stuff of egg white and also the most abundant protein in blood plasma. Albumin controls fluid distribution in the body, but researchers can borrow it’s genetic controls to mass-produce selected proteins.
Sangamo Therapeutics is using genome editing to hitch the gene for the clotting factor that’s missing in hemophilia B (factor IX, aka FIX) to the controls of the albumin gene, rather than manipulate the FIX gene directly. And instead of using the media darling CRISPR-Cas9, it relies on an older, perhaps more controllable editing tool, zinc finger nucleases (ZFNs). The strategy works in mice, and a clinical trial is just getting underway, with FDA giving fast track designation to the company’s “SB-FIX in vivo genome editing treatment for hemophilia B.”
 “Our ultimate goal is to treat children young enough so they will have a solution that will last for a lifetime and they will not have to face the consequences of the disease,” said Sandy Macrae, president and CEO of Sangamo. He calls the technology “beautiful Star Trek type science.”
Normally, in response to a puncture injury, the body stanches blood flow by knitting long, feathery strands of the protein fibrin into clots as the culmination of a cascade of interacting proteins, the clotting factors. FIX and factor VIII join in midway through the action. About 16,000 people in the U.S. have hemophilia A (factor VIII deficiency) and 4,000 have hemophilia B. Both genes are on the X chromosome, so males are affected and women are carriers.

From transfusions to gene therapy

I interviewed the first person to receive gene therapy for hemophilia A, in 2000. Born in 1949, Don Miller was raising sheep when he became the first participant in a clinical trial at the University of Pittsburgh, receiving the retrovirus-bound factor VIII gene on June 1, 1999. His story chronicles the evolution of hemophilia treatment. It began with nearly bleeding to death following his circumcision as a newborn.

Ryan White

“When I was 3, I fell out of my crib and I was black and blue from my waist to the top of my head. One time I fell at my grandmother’s house and had a 1-inch-long cut on the back of my leg. It took five weeks to stop bleeding, just leaking real slowly,” he told me. After that he limited activities and had frequent transfusions – those were the days before AIDS, which would infect 90% of the people with hemophilia before screening blood donations began. Ryan White was a teen with hemophilia A who drew attention to the vulnerability of the hemophilia community and ended use of the word “hemophiliac.”
By the time Miller had married, in 1969, he was receiving gamma globulin pooled from donated plasma and frozen, pooled factor VIII, called “cryoprecipitate.” He got into the clinical trial because he’d managed to avoid the HIV infection that kept many people with hemophilia out: “I lucked out.”
It’s just one case, but Don Miller improved – he needed boosters of factor VIII (now made more safely using recombinant DNA technology) less frequently, he had no more spontaneous bleeding, and nosebleeds stopped within minutes – as they would for anyone with normal clotting.
Alas Mr. Miller’s good response had exquisitely poor timing, for 1999 was a tragic year for gene therapy. On September 17, 1999, the death of 18-year-old Jesse Gelsinger from an overwhelming immune response to gene therapy to treat a urea cycle disorder paralyzed the field. Also that year the first boys received gene therapy for an immune deficiency that used a retrovirus, which would cause leukemia two years later, again paralyzing the field.

Clotting factor IX

But gene therapy re-emerged, with the hemophilias at the forefront. Ongoing hemophilia B clinical trials include those sponsored by UniQure Biopharma and St. Jude’s Children’s Hospital. Spark Therapeutics, in collaboration with Pfizer, recently announced preliminary data from 10 patients that showed persistent and sustained FIX production, although some patients needed steroids to quell a change in liver enzymes that could indicate an immune response. But on May 10, Dimension Therapeutics halted its trial for hemophilia B, citing “inability to achieve a minimum target product profile.” Sources tell me off-the-record that this means elevated liver enzymes that could signal an immune response.

Enter zinc fingers

Treating hemophilia B at the protein level costs $100,000 a year for “bleeds” and up to $250,000 if given two or three times a week to prevent bleeding. That’s about $20 million over a lifetime. Gene therapy seems a great alternative, a possibly one-time fix. The gene is small and only a small increase in the level of the clotting factor in plasma can make a big difference in how a patient feels.
A report from 2014 charts the “Long-Term Safety and Efficacy of Factor IX Gene Therapy in Hemophilia B,” but the vector, a type of adeno-associated virus, deposits the FIX gene in an episome, which is a bit of DNA outside of a chromosome. Like peeing in the ocean, a gene added in an episome becomes diluted as the chromosomes duplicate as cells divide, but the episomes do not. Still, gene therapy might provide enough factor IX to help adults. But young children might be a different story. For them, genome editing might be a longer-lasting approach because it inserts the healing gene at a selected site in a chromosome.

A gene therapy or genome editing approach to hemophilia would eliminate the need for frequent infusions of clotting factor.

All three genome editing technologies – CRISPR-Cas9, TALENs, and ZFNS – borrow from bacteria to create double-stranded breaks in DNA and then heal the damage. Unlike CRISPRs, which use RNA “guides,” ZFN deploys proteins, which are much more targeted and less likely to insert willy-nilly into undesired parts of the genome.
A zinc finger is a small protein that folds into an oblong shape when it binds zinc atoms, and it binds to a specific sequence of DNA found in about 3 percent of all human genes. A DNA-cutting enzyme attracted to a bound zinc finger forms a scissor-like molecular tool.
The power of zinc fingers comes from their natural affinity for transcription factor genes, which in turn encode proteins that control which suites of genes are turned on or off in a particular place and time. Zinc fingers were discovered in the African clawed frog in 1985, and first used as genetic switches in 1994.
Macrae explains how it works:

“We’ve designed zinc fingers to recognize intron 2 of the albumin gene, land on the DNA, and the restriction enzyme attaches, dimerizes, and cuts the DNA. At the same time as the ZFN cuts, we introduce a separate AAV with a good copy of the gene that encodes the missing protein.”

Some of the targeted liver cells take up the cargo and then release factor IX, which makes its way to various tissues.
He calls use of the albumin gene a “’safe harbor’ because you don’t need all of the albumin you produce. It has nice introns where we can land it and we will never remove but a fraction of the albumin genes from producing albumin.”
Meanwhile, he and his colleagues at The company is also launching clinical trials to tackle two mucopolysaccharidoses (Hunter and Hurler syndromes), and has treated 100 people with HIV with zinc fingers targeted to the CCR5 gene. ZFNs can hit any target in the genome efficiently. “We think zinc fingers is the leading genome editing technology. But eventually there will be others, and CRISPR will sort out its problems,” Macrae said.Sangamo are looking forward to the day when zinc fingers can help the youngest patients with hemophilia B:

“Parents whose children have hemophilia B came to see us, and they talked about holding their babies down to find a vein in the scalp to give an infusion. Some patients need clotting factor on a daily basis, and getting it dominates their lives. Patients die. We see what we do as part of the journey to eventually offer patients a chance to address the disease in a fundamental way.”

Podcast - Rare Diseases - Sandy Macrae

Interesting half hour recap.
https://player.fm/series/rarecast/sangamo-advances-gene-therapies-for-multiple-rare-diseases-into-the-clinic