Papers by Jeffrey C. Miller
Abstract 6563: PSMA-based detection and global proteome-based analysis of circulating tumor cells in castration resistant prostate cancer patients
Cancer research, Mar 22, 2024
Abstract 6078: Circulating tumor cell-based early detection of breast cancer
Cancer research, Mar 22, 2024
Nature communications, Feb 15, 2024
Nucleobase editors represent an emerging technology that enables precise single-base edits to the... more Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNAguided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddA tox , that can act upon double-stranded DNA. Here, we target DddA tox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddA tox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.
Enhancing gene editing specificity by attenuating DNA cleavage kinetics
Nature Biotechnology, Jul 29, 2019
Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genom... more Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.Engineered zinc finger nucleases achieve 98% knockout of a target gene in human T cells without detectable off-target activity.
Transcriptional activation of<i>Brassica napus</i>β-ketoacyl-ACP synthase II with an engineered zinc finger protein transcription factor
Plant Biotechnology Journal, Apr 23, 2012
Targeted gene regulation via designed transcription factors has great potential for precise pheno... more Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola β-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;selectable marker only&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species.
Plant Molecular Biology, Dec 27, 2008
Targeted transgene integration in plants remains a significant technical challenge for both basic... more Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and sitespecific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.
“Designer” Cells for Cellular Immunotherapy: Zinc Finger Nuclease (ZFN) Stimulated Targeted Recombination for the Simultaneous Disruption of the Endogenous Glucocorticoid Receptor and Site-Specific Addition of the IL13-Zetakine
Blood, Nov 16, 2006
Immunotherapy utilizing genetically-modified cytolytic T-lymphocytes (CTL) provides a promising t... more Immunotherapy utilizing genetically-modified cytolytic T-lymphocytes (CTL) provides a promising therapeutic approach for treating a variety of diseases, including cancer. We have demonstrated that CTLs encoding a chimeric T-cell receptor (IL13-zetakine), consisting of an extracellular IL-13 domain and a cytoplasmic CD3 domain, can be re-directed to target malignant glioma both in vitro and in animal models. This chimera re-targets the antigen-specific effector functions of modified CTLs to recognize glioblastomas due to the high expression of IL13R in these tumors. However, practical application of this approach is limited by the fact that patients undergoing surgical resection of the tumor often require treatment with glucocorticoids to control the resulting inflammation. Such treatment blocks the activity of the re-directed CTL clones and thus inhibits their therapeutic action. To overcome this limitation and render these tumor-specific CTLs resistant to glucocorticoids we have chosen to employ engineered ZFNs to specifically disrupt the endogenous glucocorticoid receptor (GR) gene. Heterodimeric ZFNs, consisting of the cleavage domain of the restriction enzyme FokI linked to engineered zinc finger DNA-binding domains, can be designed to specifically cleave a predetermined site in the genome. We have shown that these ZFN-induced double strand breaks can promote homologous recombination with high efficiency. In the present study we have investigated the use of ZFNs to simultaneously effect functional inactivation of human GR via specifically targeting the integration of the IL13-zetakine expression cassette into the GR locus itself. We can show that GR-specific ZFNs cleave their intended target sequences with high specificity and efficiency - resulting in the disruption of GR and the creation of glucocorticoid resistant cells. Moreover, we can demonstrate that these ZFNs coupled with an appropriate IL13-zetakine containing donor-DNA molecule can stimulate the integration of this chimeric T-cell receptor directly into the GR locus. Thus, this procedure results in the simultaneous knockout of GR and addition of the IL13-zetakine in a genetically defined manner. These data support the notion that ZFN-modified cells can be engineered to express chimeric antigen receptors from a predetermined genomic locus and may provide a general approach to generating effective cellular immunotherapy strategies.
Gene Correction Therapy Using Designed Zinc Finger-Based Endonucleases
Molecular Therapy, May 1, 2004
Human monogenic disorders, such as X-linked SCID, cystic fibrosis, sickle cell anemia, and hemoph... more Human monogenic disorders, such as X-linked SCID, cystic fibrosis, sickle cell anemia, and hemophilia, are caused by hypomorphic or null mutations in a single gene, and restoration of even a single wild-type allele in a sub-population of cells can be curative. In practice, however, resistance to homologous recombination (HR) is a universal feature of mammalian genomes. An important exception to
Nucleic Acids Research, Jun 8, 2010
We previously demonstrated high-frequency, targeted DNA addition mediated by the homologydirected... more We previously demonstrated high-frequency, targeted DNA addition mediated by the homologydirected DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two $750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5-10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5 0 overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5 0 overhangs on the donor DNA. Finally, we extend this nonhomologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.
738. Towards Gene Correction of X-Linked SCID Using Engineered Zinc Finger Nucleases and Integrase Defective Lentiviral Delivery
Molecular Therapy, 2006
An alternative approach to gene replacement is correction of the endogenous gene using engineered... more An alternative approach to gene replacement is correction of the endogenous gene using engineered zinc finger protein-based nucleases (ZFNs) to specifically target a DNA double stranded break at the mutation site. One mechanism the cell uses to repair these breaks is homologous ...
Nucleic Acids Research, Apr 30, 2018
Protein engineering is used to generate novel protein folds and assemblages, to impart new proper... more Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein-protein interactions, modifying protein-DNA interactions is more difficult. This may be related to the structural features of protein-DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein-DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.
Proceedings of the National Academy of Sciences of the United States of America, Apr 6, 2011
PLOS Pathogens, Apr 14, 2011
HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors ... more HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5D32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5D32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.
Nucleic Acids Research, May 29, 2008
The selective degradation of mutated mitochondrial DNA (mtDNA) molecules is a potential strategy ... more The selective degradation of mutated mitochondrial DNA (mtDNA) molecules is a potential strategy to re-populate cells with wild-type (wt) mtDNA molecules and thereby alleviate the defective mitochondrial function that underlies mtDNA diseases. Zinc finger nucleases (ZFNs), which are nucleases conjugated to a zinc-finger peptide (ZFP) engineered to bind a specific DNA sequence, could be useful for the selective degradation of particular mtDNA sequences. Typically, pairs of complementary ZFNs are used that heterodimerize on the target DNA sequence; however, conventional ZFNs were ineffective in our system. To overcome this, we created single-chain ZFNs by conjugating two FokI nuclease domains, connected by a flexible linker, to a ZFP with an N-terminal mitochondrial targeting sequence. Here we show that these ZFNs are efficiently transported into mitochondria in cells and bind mtDNA in a sequence-specific manner discriminating between two 12-bp long sequences that differ by a single base pair. Due to their selective binding they cleave dsDNA at predicted sites adjacent to the mutation. When expressed in heteroplasmic cells containing a mixture of mutated and wt mtDNA these ZFNs selectively degrade mutated mtDNA, thereby increasing the proportion of wt mtDNA molecules in the cell. Therefore, mitochondria-targeted single-chain ZFNs are a promising candidate approach for the treatment of mtDNA diseases.
Proceedings of the National Academy of Sciences of the United States of America, Apr 15, 2008
Nature Methods, Dec 5, 2010
PMC, May 1, 2010
Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on h... more Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a ''safe harbor'' locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.
A Potential Therapy for Beta-Thalassemia (ST-400) and Sickle Cell Disease (BIVV003)
Blood, Dec 7, 2017
Background: Beta-thalassemia and sickle cell disease are genetic disorders caused by mutations in... more Background: Beta-thalassemia and sickle cell disease are genetic disorders caused by mutations in the beta-globin gene which lead to significant anemia and serious medical complications. Increases in fetal hemoglobin (HbF) have been linked to improved clinical outcomes in patients with beta-thalassemia (BT) and sickle cell disease (SCD). Methods: We have developed engineered zinc finger nucleases (ZFNs) that precisely cleave and disrupt the erythroid enhancer of the BCL11A gene, which substantially boosts HbF production in erythroid progeny of genome-edited CD34 + hematopoietic stem/progenitor cells (HSPCs). The autologous modified HSPC drug product are named ST-400 (BT) and BIVV003 (SCD). Results: We demonstrate that ST-400 / BIVV003 can be manufactured by reproducible, high-level, ZFN-driven modification in peripheral blood mobilized HSPCs at clinical production scale (>10 8 cells). The drug product is made in a GMP-compliant setting using a clinical-grade electroporation device to deliver the ZFN mRNAs ex vivo . Unbiased specificity studies of ST-400 / BIVV003 demonstrated an exquisite amount of specificity, with high levels of on-target modification (~80%). Erythroid colony genotyping in enhancer targeted cells, showed bi-allelic modification of the BCL11A erythroid enhancer in >50% of HSPCs, resulting in >4-fold higher levels of gamma globin mRNA and protein compared to controls. Similarly, we observed high levels of modification in research-scale preparations of HSPCs from patients with beta-thalassemia. Injection of ST-400 / BIVV003 into immune-deficient mice resulted in robust long-term (19 week) engraftment. Targeted gene modification was maintained through multi-lineage differentiation in the bone marrow and peripheral blood. Conclusions: These results support further clinical development of ST-400 / BIVV003 as a potential therapy for beta-thalassemia and sickle cell disease. Disclosures No relevant conflicts of interest to declare.
Blood, Nov 7, 2013
• AAV delivery of ZFNs and corrective Donor vectors to adult mouse liver results in stable human ... more • AAV delivery of ZFNs and corrective Donor vectors to adult mouse liver results in stable human factor IX levels, normalizing hemophilic clotting times. Monogenic diseases, including hemophilia, represent ideal targets for genome-editing approaches aimed at correcting a defective gene. Here we report that systemic adenoassociated virus (AAV) vector delivery of zinc finger nucleases (ZFNs) and corrective donor template to the predominantly quiescent livers of adult mice enables production of high levels of human factor IX in a murine model of hemophilia B. Further, we show that off-target cleavage can be substantially reduced while maintaining robust editing by using obligate heterodimeric ZFNs engineered to minimize unwanted cleavage attributable to homodimerization of the ZFNs. These results broaden the therapeutic potential of AAV/ZFN-mediated genome editing in the liver and could expand this strategy to other nonreplicating cell types. (Blood. 2013;122(19):3283-3287)
Blood, Oct 8, 2015
AAV-and ZFN-mediated targeting of the albumin locus corrects disease phenotype in mouse models of... more AAV-and ZFN-mediated targeting of the albumin locus corrects disease phenotype in mouse models of hemophilia A and B. • Robust expression from the albumin locus provides a versatile platform for liverdirected protein replacement therapy. Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) -mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases. (Blood
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Papers by Jeffrey C. Miller