Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences

Morgan, Richard A.; Ma, Feiyang; Unti, Mildred J; Brown, Devin; Ayoub, Paul; Tam, Curtis; Lathrop, Lindsay; Aleshe, Bamidele; Kurita, Ryo; Nakamura, Yukio; Senadheera, Shantha; Wong, Randall; Hollis, Roger P.; Pellegrini, Matteo; Kohn, Donald B.

doi:10.1016/j.omtm.2020.04.006

Cited by 10 publications

(7 citation statements)

References 70 publications

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“…However, we and others have observed that LV titers and infectivity decrease with increasing proviral length, resulting in less efficient transduction of patient cells as well as increased costs for clinical and commercial applications. Optimizing the expression cassette is one strategy to create LVs with optimal titer, infectivity, and expression 5,6 but may be a prolonged process and have to be applied to each individual LV. Improvements of the vector packaging platform or the manufacturing protocol can provide a global solution to the production of many different LVs.…”

Section: Introductionmentioning

confidence: 99%

Improved lentiviral vector titers from a multi-gene knockout packaging line

Han¹,

Tam²,

Tam³

et al. 2021

Molecular Therapy - Oncolytics

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Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2 0 -5 0 -oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to $2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded $7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by $2fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to $11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.

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Section: Introductionmentioning

confidence: 99%

Improved lentiviral vector titers from a multi-gene knockout packaging line

Han¹,

Tam²,

Tam³

et al. 2021

Molecular Therapy - Oncolytics

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“…Our rAAV-based approach enabled efficient transduction of the mammalian central nervous system. Our approach differs from recent MPRA strategies which use lentiviral vectors 29,[61][62][63] in that AAV is expressed episomally while lentivirus must first integrate into the genome. Lentivirus is especially useful for in vitro applications with difficult to transfect cell types, as AAV is not very efficient at transducing cell lines 64 .…”

Section: Discussionmentioning

confidence: 99%

Parallel functional testing identifies enhancers active in early postnatal mouse brain

Lambert

Su-Feher

Cichewicz

et al. 2021

eLife

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Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.

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“…MPRAs are becoming widely used as an effective tool to assess functionality of cis -regulatory DNA elements in a high-throughput manner ( Ernst et al, 2016 ; Rabani et al, 2017 ; Mattioli et al, 2019 ; Shigaki et al, 2019 ; Choi et al, 2020 ; Davis et al, 2020 ; Ireland et al, 2020 ; King et al, 2020 ; Klein et al, 2020 ; Morgan et al, 2020 ; Renganaath et al, 2020 ). In addition, several modifications to the approach have been described that broaden its applicability ( Rosenberg et al, 2015 ; Shen et al, 2016 ; Safra et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

MPRAdecoder: Processing of the Raw MPRA Data With a priori Unknown Sequences of the Region of Interest and Associated Barcodes

et al. 2021

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Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of numerous DNA regulatory elements and/or their mutant variants. The assays are based on the construction of reporter plasmid libraries containing two variable parts, a region of interest (ROI) and a barcode (BC), located outside and within the transcription unit, respectively. Importantly, each plasmid molecule in a such a highly diverse library is characterized by a unique BC–ROI association. The reporter constructs are delivered to target cells and expression of BCs at the transcript level is assayed by RT-PCR followed by next-generation sequencing (NGS). The obtained values are normalized to the abundance of BCs in the plasmid DNA sample. Altogether, this allows evaluating the regulatory potential of the associated ROI sequences. However, depending on the MPRA library construction design, the BC and ROI sequences as well as their associations can be a priori unknown. In such a case, the BC and ROI sequences, their possible mutant variants, and unambiguous BC–ROI associations have to be identified, whereas all uncertain cases have to be excluded from the analysis. Besides the preparation of additional “mapping” samples for NGS, this also requires specific bioinformatics tools. Here, we present a pipeline for processing raw MPRA data obtained by NGS for reporter construct libraries with a priori unknown sequences of BCs and ROIs. The pipeline robustly identifies unambiguous (so-called genuine) BCs and ROIs associated with them, calculates the normalized expression level for each BC and the averaged values for each ROI, and provides a graphical visualization of the processed data.

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Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences

Cited by 10 publications

References 70 publications

Improved lentiviral vector titers from a multi-gene knockout packaging line

Improved lentiviral vector titers from a multi-gene knockout packaging line

Parallel functional testing identifies enhancers active in early postnatal mouse brain

MPRAdecoder: Processing of the Raw MPRA Data With a priori Unknown Sequences of the Region of Interest and Associated Barcodes

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