OUR ADVANCED PLATFORM FOR GENOMIC MEDICINE

Treating serious diseases by correcting mistakes in DNA has the potential to open a promising new frontier of genomic medicines. These potential new therapeutic candidates must be designed to the highest standards of precision, efficiency and specificity.

As a pioneer and leader of therapeutic genome editing, Sangamo has developed an advanced platform for developing genomic medicines. Based on natural human proteins that recognize specific DNA sequences, Sangamo’s zinc finger protein technology is designed for:

  • Single-nucleotide precision
    High design density means zinc finger-based therapeutic candidates can target almost any sequence in the genome, enabling development of treatments for technically challenging genetic conditions
  • High efficiency 
    Our therapeutic ZFNs candidates can achieve almost complete editing of their targeted locus
  • Extreme specificity
    ZFNs are engineered to engage the target site with almost no detectable off-target effects

What are zinc fingers?

Every cell in our body contains the same genes. Yet neurons, muscle cells, and skin cells look and work differently. To specialize, our cells turn certain genes on or off when needed.

Naturally occurring zinc finger proteins enable our cells to pinpoint a gene among thousands of others and to regulate its expression. They are named for how they use a zinc atom to fold into a compact shape that can fit inside the major groove of DNA, enabling recognition of specific base sequences.

As the most abundant DNA binding protein in humans, thousands of naturally occurring zinc finger proteins exist to recognize and bind specific DNA sequences, with each finger binding three DNA nucleotides or “bases” at a time.

Shift Target Sequence

In the floor of the major groove, the base edges of a given sequence present a unique and complex molecular landscape that is specific for each sequence. Shifting by even a single base pair presents a dramatically altered pattern of bases that must be recognized, which will require an entirely different set of zinc fingers.

Sangamo uses a library of more than 3,200 zinc finger modules to engineer potential therapeutic tools for targeting a specific location in the human genome. By attaching different functional domains to those location-specific arrays of zinc fingers, or zinc finger proteins, Sangamo is able to edit and repair DNA, or adjust the expression level of a particular gene to treat serious diseases.

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Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) enable genome editing, which may be designed to delete a certain gene or sequence of DNA and/or add a new, therapeutic gene or sequence of DNA at a precise location.

ZFNs are engineered by attaching a nuclease, a DNA cutting enzyme, to a pair of zinc finger proteins. A pair of ZFNs is used to precisely and specifically cut and edit DNA with very high efficiency -- like a pair of molecular scissors.

Zinc Finger Protein Transcription Factors

Zinc finger protein transcription factors (ZFP-TFs) are used to regulate gene expression by either activating or repressing the activity of a gene or an individual gene allele.

ZFP-TFs are engineered by attaching an activation or repression domain to a designed zinc finger protein. Unlike ZFNs, only one ZFP-TF is needed to regulate the activity of a gene.

A key feature of a ZFP-TF is its potential to differentiate between the wild-type and mutant allele of a gene, enabling the treatment of certain genetic diseases in which only one copy of a gene is problematic.

Zinc Finger Protein Transcription Factors (ZFP-TFs)

Of the many approaches to reduce tau expression that we've studied, zinc finger protein gene regulation technology is especially promising for its exquisite specificity, its potent reduction of tau protein expression, and its potential to provide a durable, long-lasting effect with only a single administration.

- Bradley Hyman, MD, PhD

  Director, Massachusetts Alzheimer's Disease Research Center
  Alzheimer's Unit Director, MassGeneral Institute for Neurodegenerative Disease
  Professor of Neurology, Harvard Medical School

PRECISION TO TARGET ANY LOCATION IN THE GENOME

Hundreds of active ZFN leads that precisely cut a nucleotide within a target DNA sequence can be designed, assembled and validated in 10 days. Based on these characteristics, zinc fingers have the potential to be the ideal gene editing platform to treat challenging genetic diseases.

Comparing ZFN and CRISPR/Cas9* Precision

Compared to other gene editing platforms, ZFNs can precisely target more sites within a given region of DNA, as shown here for a therapeutically critical sequence within the beta-globin gene HBB.

* High fidelity CRISPR/Cas9 with a five prime Guanine restriction in the guide.

Precision is crucial when developing gene editing therapies. It's important to cut exactly at the mutated base, or just next to it. Cutting even a few bases away may significantly reduce the probability of correcting or removing the mutation, due to how our cells naturally repair DNA.

For example, a form of blindness called Leber’s congenital amaurosis is caused by a single base mutation, named LCA10, within the CEP290 gene. To protect a person’s vision, this mutation must be removed from the gene in as many photoreceptors as possible. A high targeting density enables hundreds of designs that cleave into the LCA10 mutation.

Targeting the LCA10 Mutation in the CEP290 Gene with ZFNs

The modularity of the zinc finger platform generates hundreds of ZFN options for the DNA sequence surrounding the LCA10 mutation, with multiple ZFN designs precisely targeting the mutated base.

This high design density allows Sangamo to choose the optimal ZFN pair for correcting the LCA10 mutation and rescuing the greatest number of photoreceptors.

604
ZFN Design Options

THE LATEST ENHANCEMENTS TO THE ZFN PLATFORM

Sangamo built a substantial library of more than 3,000 zinc finger modules over the last decade. The latest enhancements focus on other aspects, such as:

New linkers for skipping one or two bases to enable us to address a much wider range of targets, with consequent improvements in activity and specificity.

THE LATEST ENHANCEMENTS TO THE ZFN PLATFORM

Sangamo built a substantial library of more than 3,000 zinc finger modules over the last decade. The latest enhancements focus on other aspects, such as:

New dimer configurations, in which the nucleases are attached to the zinc finger proteins at either the C or N-terminus, effectively quadrupling the possible ZFN dimer configurations around a cut site, enabling an unprecedented flexibility to target even the most challenging locations in the genome.

EXCEPTIONAL EFFICIENCY

A gene editing therapy’s ability to correctly edit a large number of a patient’s cells is an important component of its value. Zinc finger nucleases (ZFNs) are able to cut with extremely high efficiency across various cell types.

High ZFN Editing Efficiency Across Gamma Globin Promoter for HPFH Mutations

Initial ZFN leads can efficiently edit at nearly every nucleotide in the region of the gamma globin promoter, spanning key mutations for hereditary persistence of fetal hemoglobin. Any missed base location can be edited efficiently with optimized ZFN designs.

IN VIVO GENE EDITING THERAPY CANDIDATES

Over the past decade, Sangamo has continuously improved the efficiency of the platform such that ZFNs are now able to edit almost any human cell type.

In the context of in vivo gene editing, in which DNA editing occurs inside the body, an important factor of efficiency for any gene editing tool is adequate delivery to the target tissue or cell type.

There are several delivery methods currently being developed, including viral approaches using adeno-associated virus (AAV) vectors, and non-viral approaches, such as lipid nanoparticles (LNP). Recent work at Sangamo has focused on improving these delivery vehicles to maximize the concentration of ZFNs inside the target cell in order to efficiently perform the editing.

AAV Delivery

LNP Delivery

EX VIVO GENE EDITING FOR CELL THERAPY CANDIDATES

For gene-edited cell therapies, efficiency depends upon both optimized delivery methods and careful treatment and processing of specific types of cells. In these therapy candidates, cells can be taken from a patient (autologous) or a healthy donor (allogeneic) and transferred to a cGMP manufacturing facility, where one or multiple genes can be edited simultaneously while carefully maintaining the cells’ health. The edited cells are then transfused or transplanted into a patient.

Sangamo is currently developing gene-edited cell therapy candidates for beta thalassemia, sickle cell disease, various forms of cancer and immunological diseases. These programs draw upon years of experience in the manufacturing of cell therapy candidates for its T-cell and hematopoietic stem cell (HSC) programs in HIV that were the first ex vivo gene-edited cell therapy candidates to enter human clinical trials.

76%

of cells have all 4 edits

Efficient Multiplex Editing for Next-Generation Universal T-Cell Therapy Candidates

The unmatched multiplex gene editing efficiency of ZFNs enables development of next-generation allogeneic CAR-T and engineered T-cell therapy candidates with the potential ability to combat cancer and other challenging diseases.

UNMATCHED SPECIFICITY

Many genes share similar DNA code. Genome editing therapeutic candidates therefore must be highly specific to avoid binding and altering healthy DNA. The following aspects of the ZFN genome editing platform enable designing zinc finger nuclease-based therapeutic candidates with almost no detectable off-target activity.

Protein engineering – Any non-specific contacts between DNA and ZFN or ZFP-TF can be systemically removed. This reduces off-target activity by up to 3000-fold, producing ZFN designs with undetectable off-target activity using the most stringent tests available (oligo-capture assay).

On-Target Activity

82.8
%

Off-Target Activity

Ablating Non-specific Contacts to DNA Backbone Eliminates Detectable Off-Target Activity

Many genes share similar DNA code. Genome editing therapeutic candidates therefore must be highly specific to avoid binding and altering healthy DNA. The following aspects of the ZFN genome editing platform enable designing zinc finger nuclease-based therapeutic candidates with almost no detectable off-target activity.

Ability to target any base – Due to ZFNs’ high design density, their unique ability to target any base pair in the genome allows selecting the safest locations for editing. Additionally, the same cut site can be targeted by multiple ZFN designs that have different binding profiles, preventing off-target editing of pseudogenes and homologous DNA sequences elsewhere in the genome.

Many genes share similar DNA code. Genome editing therapeutic candidates therefore must be highly specific to avoid binding and altering healthy DNA. The following aspects of the ZFN genome editing platform enable designing zinc finger nuclease-based therapeutic candidates with almost no detectable off-target activity.

Targeting long sequences of DNA – A pair of ZFNs targets sequences of typically 30-36 base pairs, as it’s unlikely for an identical sequence of that length to recur elsewhere in the genome. This also protects against unanticipated off-target events due to single nucleotide polymorphisms (natural DNA variations between people).

36
bps

Develop the Future of Genomic Medicine

Our zinc finger technology provides an ideal platform to develop genome editing and gene regulation therapy candidates. We collaborate to harness the capabilities and competitive advantages of industry partners for product candidates that require expertise or resources outside our areas of focus.