Clinical use of lentiviral vectors

Abstract

Viral vectors provide an efficient means for modification of eukaryotic cells, and their use is now commonplace in academic laboratories and industry for both research and clinical gene therapy applications. Lentiviral vectors, derived from the human immunodeficiency virus, have been extensively investigated and optimized over the past two decades. Third-generation, self-inactivating lentiviral vectors have recently been used in multiple clinical trials to introduce genes into hematopoietic stem cells to correct primary immunodeficiencies and hemoglobinopathies. These vectors have also been used to introduce genes into mature T cells to generate immunity to cancer through the delivery of chimeric antigen receptors (CARs) or cloned T-cell receptors. CAR T-cell therapies engineered using lentiviral vectors have demonstrated noteworthy clinical success in patients with B-cell malignancies leading to regulatory approval of the first genetically engineered cellular therapy using lentiviral vectors. In this review, we discuss several aspects of lentiviral vectors that will be of interest to clinicians, including an overview of lentiviral vector development, the current uses of viral vectors as therapy for primary immunodeficiencies and cancers, large-scale manufacturing of lentiviral vectors, and long-term follow-up of patients treated with gene therapy products.

Fig. 1

Steps of reverse transcription. This figure illustrates the steps involved in the conversion of the single-stranded RNA (ssRNA) genome of HIV into double-stranded DNA (dsDNA). RNA is shown in red and DNA as yellow [1]. The transfer RNA (tRNA) primer (blue ellipse) is base paired to the primer-binding site (PBS) [2]. Reverse transcription is initiated using the reverse transcriptase (RT; purple ellipse) enzyme, and minus-strand DNA synthesis starts from the tRNA primer, copying the U5 and R sequences at the 5′ end of the genome. At this stage, an RNA/DNA duplex is created, and ribonuclease (RNase) H activity of the RT enzyme degrades the viral RNA that has been copied (dotted red line) [3, 4]. The minus-strand DNA has been transferred, using the R sequence found at both ends of the viral RNA, to the 3′ end of the viral RNA, and minus-strand DNA synthesis continues. The HIV-1 genome has two RNase H-resistant polypurine tracts (PPTs) [5]. The two PPTs serve as primers for plus-strand DNA synthesis. One plus-strand is initiated at U3, and one is initiated at the central PPT (cPPT) [6]. Both the plus- and minus-strand DNAs are then elongated, finally resulting in a complete copy of viral RNA with additional sequences at the 5′ and 3′ ends such that viral DNA has an identical copy of U3RU5 at both ends. The plus-strand that was initiated at U3 displaces a segment of the plus-strand that was initiated from the cPPT, creating a small flap called the central flap (cFLAP). LTR long terminal repeat

Fig. 2

Third-generation lentiviral vector. Third-generation lentiviral vectors are composed of two separate packaging plasmids, one encoding gag and pol and another encoding rev. An additional plasmid encodes the envelope protein, derived from the VSV-G. The plasmid encoding the gene of interest contains lentiviral LTR sequences that have been altered to be self-inactivating (SIN) to prevent recombination. LTR long terminal repeat, VSV vesicular stomatitis virus

Fig. 3

Overview of large-scale vector manufacturing. Manufacture of a lentiviral vector begins with the culture of a packaging cell line in a facility that uses Good Manufacturing Practices. The cells are transfected with the plasmids that make up the third-generation lentiviral vector, and the vector-producing cells are expanded in culture. The vector is purified from the cells and culture debris and filtered to ensure sterility, and individual aliquots are cryopreserved

Fig. 4

Key clinical uses of lentiviral vectors. a Correction of primary immunodeficiency. Using a viral vector to deliver the common gamma chain (γc) restores immune function in patients with SCID-X1. b Delivery of a tumor-specific T-cell receptor (TCR). A lentiviral vector can be used to introduce the MART-1 TCR, which recognizes a melanoma antigen, into a patient’s T cells ex vivo. The modified T cells, which now recognize melanoma cells, are administered to the patient as a cancer therapy. c Chimeric antigen receptor (CAR) T-cell therapy. A CAR engineered from three distinct domains (antigen recognition, co-stimulatory signaling, and T-cell signaling) can be introduced into T cells using a lentiviral vector. The cells expressing the modified receptor recognize the antigen of interest and harness the potent cytotoxic activity of T cells to attack tumor cells. Currently, most CAR T-cell therapies in clinical trials target the CD19 antigen, a protein expressed on B cells and B-cell malignancies. SCID severe combined immunodeficiency