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"Kim, Kenneth A"
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Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery
Achieving the full potential of zinc-finger nucleases (ZFNs) for genome engineering in human cells requires their efficient delivery to the relevant cell types. Here we exploited the infectivity of integrase-defective lentiviral vectors (IDLV) to express ZFNs and provide the template DNA for gene correction in different cell types. IDLV-mediated delivery supported high rates (13–39%) of editing at the IL-2 receptor common γ-chain gene (
IL2RG
) across different cell types. IDLVs also mediated site-specific gene addition by a process that required ZFN cleavage and homologous template DNA, thus establishing a platform that can target the insertion of transgenes into a predetermined genomic site. Using IDLV delivery and ZFNs targeting distinct loci, we observed high levels of gene addition (up to 50%) in a panel of human cell lines, as well as human embryonic stem cells (5%), allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.
Journal Article
An improved zinc-finger nuclease architecture for highly specific genome editing
Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.
Journal Article
Engineering HIV-Resistant Human CD4+ T Cells with CXCR4-Specific Zinc-Finger Nucleases
HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 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 ccr5Δ32 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.
Journal Article
Preclinical development and qualification of ZFN-mediated CCR5 disruption in human hematopoietic stem/progenitor cells
Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC). The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinical
and
testing to demonstrate the feasibility and (preclinical) safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potential
and
. Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov).
Journal Article
Corporate Culture and Investment-Cash Flow Sensitivity
Can firms overcome credit constraints with a corporate culture of high integrity? We empirically address this question by studying their investment-cash flow sensitivities. We identify firms with a culture of integrity through textual analysis of public documents in a sample of Chinese listed firms and also through corporate culture statements. Our results show that firms with an integrity-focused culture have lower investment-cash flow sensitivity, even after we address endogeneity concerns. However, we also find that for the culture to reduce the investment-cash flow sensitivity, external stakeholders must be able to verify this culture through a low information asymmetry environment. Overall, our findings show that a corporate culture of high integrity can mitigate a firm's external transaction costs.
Journal Article
Price Limit Performance: Evidence from the Tokyo Stock Exchange
Price limit advocates claim that price limits decrease stock price volatility, counter overreaction, and do not interfere with trading activity. Conversely, price limit critics claim that price limits cause higher volatility levels on subsequent days (volatility spillover hypothesis), prevent prices from efficiently reaching their equilibrium level (delayed price discovery hypothesis), and interfere with trading due to limitations imposed by price limits (trading interference hypothesis). Empirical research does not provide conclusive support for either positions. We examine the Tokyo Stock Exchange price limit system to test these hypotheses. Our evidence supports all three hypotheses suggesting that price limits may be ineffective.
Journal Article
Bank Competition and Leverage Adjustments
We test whether bank competition affects firms' leverage adjustment speeds. Using Chinese data where bank concentration varies across both years and provinces, we find that underlevered firms move to their target leverage faster when bank competition is high. Tests surrounding an exogenous shock to bank competition lead to the same conclusion. We also find that small firms and nonstate-owned firms exhibit faster leverage adjustments when bank competition is high, which is consistent with the conjecture that bank risk taking increases with competition.
Journal Article
Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases
Homozygosity for the naturally occurring
Δ32
deletion in the HIV co-receptor
CCR5
confers resistance to HIV-1 infection. We generated an HIV-resistant genotype
de novo
using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous
CCR5
. Transient expression of CCR5 ZFNs permanently and specifically disrupted ∼50% of
CCR5
alleles in a pool of primary human CD4
+
T cells. Genetic disruption of
CCR5
provided robust, stable and heritable protection against HIV-1 infection
in vitro
and
in vivo
in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4
+
T cells had lower viral loads and higher CD4
+
T-cell counts than mice engrafted with wild-type CD4
+
T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4
+
T-cell population. Thus adoptive transfer of
ex vivo
expanded CCR5 ZFN–modified autologous CD4
+
T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.
Journal Article
Zinc-finger Nuclease Editing of Human cxcr4 Promotes HIV-1 CD4+ T Cell Resistance and Enrichment
HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4+ T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4+ T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4+ T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4+ T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4+ T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4+ T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4+ T cells during HIV-1 infection.
Journal Article