DNA binding alone is not sufficient for a transcription factor to induce a change in gene expression. A second component must also be brought into close proximity to the gene in order to regulate it. In higher organisms, transcription factors typically consist of two principal components: a DNA-binding domain and a functional domain that causes the target gene to be activated or repressed. (fig. B)

At Sangamo we engineer novel transcription factors that mimic the natural mode of gene regulation. Our technology is based on the engineering of a naturally occurring class of DNA-binding proteins, zinc finger DNA binding proteins or ZFPs. We can engineer ZFPs to recognize a DNA sequence close to or within a gene of choice. By attaching a functional domain to that ZFP we generate a ZPF transcription factor (ZFP TF) that can regulate the target gene up or down (see above) or a ZFN that can modify a gene.

Engineering ZFPs to Modify Genes
ZFPs can be engineered with a DNA-cutting, or nuclease, domain to generate zinc finger nucleases or ZFNs. The ZFN is able to "recognize" its intended gene target through its engineered (ZFP) DNA binding domain (fig. B). However, instead of regulating the expression of the target gene (as with a ZFP TF), the ZFN causes the gene to be cut near the ZFP binding site triggering a natural repair process.

Why do we use ZFPs?
ZFPs are the only DNA-binding motifs that are amenable to engineering. Each “finger” structure is a small modular unit that recognizes and binds to 3 base pairs of DNA. These modules can be joined together to bind longer DNA sequences. This diagram represents a 3-finger ZFP (in green) binding to a nine base pair long sequence of DNA (in blue). Each “finger” contains a zinc atom (pink) that maintains its structure; one particular region of the finger, known as the alpha helix (depicted in yellow on the first finger) is used to make contact with the DNA. (fig. C)

A closer view of the alpha helix shown here highlights the four key amino acid residues that make contact with the DNA. If different residues are substituted in these positions then a different sequence of DNA will be recognized and bound. Sangamo has done extensive work to determine which combinations of residues at these positions enable recognition and binding of which DNA bases. (fig. D)

DNA Binding Domain
Sangamo scientists can design and engineer ZFPs to bind to sequences in any target gene. The ZFP is attached to an appropriate functional domain to generate a ZFP TF that can regulate that gene. DNA encoding the ZFP TF or ZFN is introduced into cells. When the ZFP TF or ZFN protein is expressed, it binds to the DNA of the target gene and has a biological effect dependent on the functional domain being used. (fig. E)

Using our ZFP Technology genes can be:
Activated:
Human cells transfected with a ZFP TF designed to upregulate the expression of the human EPO gene produce more EPO protein that cells that undergo a normal physiological response to low oxygen levels. (fig. F)

Repressed:
Repressed: Human cells expressing Vascular Endothelial Growth Factor- A (VEGF-A) were stably integrated with a gene encoding a ZFP TF designed to down regulate the expression of the VEGF-A gene. The expression of the ZFP TF was regulated by tetracycline. Adding tetracycline to the cells causes increased expression of the ZFP TF which then shuts down the expression of VEGF. (fig. G)

Corrected:
Genetic diseases such as X-linked SCID and sickle cell anemia are caused by mutations within single genes. "Gene Correction" is the process by which a mutation, or disease causing DNA sequence, can be repaired with the "correct" DNA sequence, restoring normal gene function.  Our engineered ZFPs can be attached to nuclease domains to create ZFNs. The ZFN is able to "recognize" its intended gene target through its engineered (ZFP) DNA binding domain (fig. B). However, instead of regulating the expression of the target gene (as with a ZFP TF), the ZFN causes the gene to be cut near the ZFP binding site triggering a repair process and facilitating the incorporation of the corrected DNA sequence into the chromosomal location where the disease related mutation previously existed (fig. H).  A segment of DNA or “donor sequence” that encodes the correct gene sequence is also introduced into the cell to provide a template for the correction of the cellular gene. The process of gene correction occurs naturally and is called homologous recombination (HR). While gene correction has been pursued in academic research laboratories for over a decade, its clinical application has been limited by the low efficiency of HR, the biological process of gene repair. HR occurs naturally at a rate of approximately once in every one million cells receiving the DNA donor sequence; this rate is too low to be of clinical use. However, we, and our collaborators, have shown that the use of engineered ZFNs to cleave the target gene near the defective sequence can increase the efficiency of targeted HR by several thousand times. ZFP Therapeutic gene correction is a revolutionary technical approach to gene repair because ZFNs, like all ZFPs, can be engineered to recognize virtually any target gene in the human genome. We are working to generate the preclinical data necessary to evaluate the potential utility of this approach for X-linked SCID and hemoglobinopathies such as sickle cell anemia and b-Thalassemia.

Disrupted:
ZFNs can also be used to disrupt a gene sequence.  This may have therapeutic applications in diseases such as HIV and hepatitis C.  To effect ZFN-mediated gene disruption, ZFNs are introduced into cells without an added DNA donor sequence.  Under these circumstances, introduction of a double stranded break in the cellular gene prompts the cell’s repair machinery to rejoin the two broken ends of the DNA (fig. I) In so doing, this error-prone process introduces alterations into the gene sequence, disrupting the gene’s normal coding sequence.  This disruption frequently results in a shortened or non-functional protein product.  In the case of HIV we are using this approach to disrupt the gene that encodes a cellular protein, CCR5, which is an essential co-factor for HIV entry and infection of T-cells and other cells of the immune system.

We are using our ZFP technology in the following applications:

Human Therapeutics

• ZFP Therapeutics™
ZFP TFs and ZFNs have the potential to be developed as pharmaceutical products to treat a broad spectrum of diseases through the regulation or modification of disease-related genes in patients.

Enabling Technology Applications

• ZFP TF and ZFN Engineered Cell Lines for the Manufacturing of
Protein Pharmaceuticals
ZFP TF- and ZFN-engineered cell lines can be developed to enhance production yields of protein pharmaceuticals and monoclonal antibodies.

• Agricultural Biotechnology
ZFP TFs can be used to regulate genes and ZFNs to modify or disrupt genes in plants, leading to potential applications in the development of new crops for optimized yield with enhanced nutritional properties.