Transient Gene Expression by Nonintegrating Lentiviral Vectors Sarah J. Nightingale, Roger P. Hollis, Karen A. Pepper, Denise Petersen, Xiao-Jin Yu, Catherine Yang, Ingrid Bahner, Donald B. Kohn  Molecular Therapy  Volume 13, Issue 6, Pages 1121-1132 (June 2006) DOI: 10.1016/j.ymthe.2006.01.008 Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Design and construction of NIL vectors. The lentiviral vector is shown schematically in the form of plasmid and provirus. Mutations introduced by site-directed mutagenesis of the plasmid LTR are copied into the other LTR of the vector provirus during reverse transcription. R, U5, and U3 are components of the LTR. U3 regions were self-inactivating (SIN). CA in U5 and U3 represents the location of the conserved CA dinucleotide in the plasmid and provirus. Ψ ΔGag represents the encapsidation sequence. RRE is the Rev-responsive element. Two reporter genes were utilized: enhanced green fluorescent protein (eGFP) and neomycin resistance gene (neoR). Molecular Therapy 2006 13, 1121-1132DOI: (10.1016/j.ymthe.2006.01.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 2 eGFP expression from NIL vectors packaged with wild-type integrase (Int+) and defective Integrase (Int−) in Jurkat cells. Jurkat human T cells were transduced with the indicated vectors and analyzed by flow cytometry to determine the percentage of cells expressing eGFP at various times. NIL vectors had: (A) single base-pair changes in the CA dinucleotides of the LTR and wild-type integrase, (B) double base-pair changes and wild-type integrase, (C) single base pair changes and defective integrase, or (D) double base-pair changes and defective integrase. Molecular Therapy 2006 13, 1121-1132DOI: (10.1016/j.ymthe.2006.01.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 3 eGFP expression from NIL vectors packaged with wild-type integrase (Int+) and defective integrase (Int−) in primary CD34+ progenitor cells. Human CD34+ hematopoietic progenitor cells were transduced with the indicated vectors and analyzed by flow cytometry to determine the percentage of cells expressing eGFP at various times. NIL vectors had: (A) single base-pair changes in the CA dinucleotides of the LTR and wild-type integrase, (B) double base-pair changes and wild-type integrase, (C) single base-pair changes and defective integrase, or (D) double base-pair changes and defective integrase. Molecular Therapy 2006 13, 1121-1132DOI: (10.1016/j.ymthe.2006.01.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Southern blot analysis of vector DNA present over time in cells treated with wild-type and NIL vectors. Jurkat human T cells were transduced with the indicated wild-type or NIL vectors. DNA was isolated from vector-treated cells at 3, 15, and 30 days posttransduction, restriction digested with the indicated enzymes, and analyzed by Southern blot. The negative control (neg) is digested DNA from Jurkat cells that had not been treated with a vector. Molecular Therapy 2006 13, 1121-1132DOI: (10.1016/j.ymthe.2006.01.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Southern blot analysis of the forms of integrated vector present in clones of NIL vector-treated neomycin-resistant HT29 cells. HT29 human colon carcinoma cells were transduced with the indicated wild-type or NIL vectors for 24 h and were selected in G418-containing medium for 4 weeks. DNA was isolated from wild-type and NIL-vector-treated, G418-resistant HT29 clones, restriction digested with AflII, and analyzed by Southern blot. The positive control (pos) was 10 pg pCCL-SIN-CMV-neoR plasmid mixed with 10 μg untreated genomic DNA and digested with AflII. The negative control (neg) is digested DNA from HT29 cells that had not been treated with a vector. The asterisk indicates a repeat of 5′++/3′++, Int+ 2. Molecular Therapy 2006 13, 1121-1132DOI: (10.1016/j.ymthe.2006.01.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Inverse PCR approach to rescue and sequence the vector–host DNA junction. (A) HT29 human colon carcinoma cells were transduced with the indicated wild-type or NIL vectors for 24 h and were selected in G418-containing medium for 4 weeks. DNA was isolated from wild-type- and NIL-vector-treated, G418-resistant HT29 clones and digested with the restriction enzymes shown. Three restriction enzyme digestions/PCR strategies were designed to be specific for the 5′ LTR, the 3′ LTR, and both LTR together. Digested DNA was diluted to promote intramolecular ligation. The locations of primers used for inverse PCR are indicated by arrows. Inverse PCR was followed by nested PCR, which resulted in well-defined bands for sequencing. (B) The schematic diagram shows the vector with its LTR, flanked by the host chromosomal DNA. Sequence for the top strand of vector DNA flanked by arrows representing the host DNA is shown directly underneath the schematic diagram. DNA sequences from clones derived from cells treated with wild-type and NIL vectors are shown below, separated by horizontal lines. Since sequences for only the top strand of vector DNA are shown, the terminal CA dinucleotide (underlined) is visible at the 3′ end, whereas the complementary TG (underlined) is visible at the 5′ end. The dashed line (…….) indicates a deletion or rearrangement at the region of interest (lack of characteristic LTR/chromosomal junction). Sequences surrounded by a box denote vector sequences. Lowercase text represents chromosomal sequences. Bold text represents the 5-bp genomic sequence duplication. Mutated dinucleotide bases are italicized and in bold. Internal vector sequences (not shown) are indicated by an arrow. Molecular Therapy 2006 13, 1121-1132DOI: (10.1016/j.ymthe.2006.01.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions