Methods for gene transfer to the central nervous system

Abstract

Gene transfer is an increasingly utilized approach for research and clinical applications involving the central nervous system (CNS). Vectors for gene transfer can be as simple as an unmodified plasmid, but more commonly involve complex modifications to viruses to make them suitable gene delivery vehicles. This chapter will explain how tools for CNS gene transfer have been derived from naturally occurring viruses. The current capabilities of plasmid, retroviral, adeno-associated virus, adenovirus, and herpes simplex virus vectors for CNS gene delivery will be described. These include both focal and global CNS gene transfer strategies, with short- or long-term gene expression. As is described in this chapter, an important aspect of any vector is the cis-acting regulatory elements incorporated into the vector genome that control when, where, and how the transgene is expressed.

Keywords: AAV; Adenovirus; CNS; Gene therapy; Lentivirus; Plasmid; Promoter; Vector.

Figure 3.1

Recombinant AAV vector genome design. (A) The wild-type AAV genome generally consists of the viral rep and cap genes between two inverted terminal repeats (ITRs). (B) Recombinant AAV vector is produced by cotransfection of a vector plasmid containing an ITR-flanked transgene cassette, a packaging plasmid that encodes the rep and cap genes of a specific AAV serotype and a helper plasmid that supplies the essential adenovirus helper genes (E1a, E1b, E2a, E4, and VA RNA). The vector plasmid may be either single-stranded DNA that encodes ~4.5 kb of novel transgene sequence or self-complementary (sc) DNA that can encodes ~2.2 kb of novel transgene sequence in duplex form. The sc AAV genome consists of the forward and reverse complement (rc) transgene sequences with wild-type ITRs at the extremities and a mutated ITR (X) at the axis of symmetry.

Figure 3.2

Retroviridae family comprises seven major genera: alpha-retroviruses (prototype ALSV), beta-retrovirus (prototype MMTV), delta-retrovirus (prototype HTLV-I, HTLV-II), gamma-retrovirus (prototype MLV), epsilon-retrovirus (prototype WDSV), lentiretroviruses (prototype HIV-1), and spumaviruses (prototype SFV). Notable characteristics of the viruses are included.

Figure 3.3

Life cycle of the HIV-1 including binding-fusion, uncoating, reverse transcription, integration, transcription, nuclear export, assembly and virion budding are shown. The star represents reverse transcriptase. Steps of the life cycle labeled from one to eight.

Figure 3.4

Development of the packaging cassette. Four generations of the packaging cassette are shown. (A) First generation included all four accessory proteins, Vpu, Vpr, Vif, and Nef and the regulatory proteins, tat and rev. RRE stands for rev response element. Expression is driven from the CMV promoter; PolyA signal (pA) is shown. (B) Second generation excluded all four accessory proteins, Vpu, Vpr, Vif, and Nef, but included regulatory proteins, tat and rev. (C) Further split of the packaging cassette defined the third generation. (D) Four generation characterized by a further split of the cassette into three components: gag expressed from the CMV promoter; Vpr-protease expressed from the EF1-α promoter. Vpr-pol transcript is driven from the EF1-α promoter. All three packaging cassettes contain RRE and pA signals. CMV, cytomegalovirus; RRE, Rev response element.

Figure 3.5

Development of the expression (transgene) cassette. (A) First generation contains wild-typed 5′- and 3′-LTRs, primer-binding site (PBS), splice donor (SD), and splice acceptor (SA), central polypurine tract (cPPT) and polypurine tract (PPT), Rev response element (RRE), woodchuck hepatitis virus posttranscriptional regulatory element (wPRE), and the retroviral vector packaging element, psi (Y) signal. Expression of a transgene is driven from the promoter-of-choice shown by the arrows. (B) SIN vector is devoid of the parental enhancer/promoter sequences, located at the U3′ of the 3 ′-LTR (deletion is shown); thus, lacking the ability to transcribe full-length mRNA. CMV promoter incorporated in the 5′-LTR employed to generate full-length mRNA. (C) Inducible expression cassette drives from the tetracycline response element (TRE) incorporated in the U3 region of the 3′-LTR. This cassette named a conditional SIN cassette, expressing the genome in the presence of the ttA. SIN, self-inactivating; LTR, long terminal repeat; CMV, cytomegalovirus.

Figure 3.6

Transient transfection protocol employed to generate lentiviral particles. 293T cells transfected with VSV-G, packaging, and transgene cassettes. Viral particles that bud out from the cell membrane contain full-length RNA of the vector (expressed from the transgene cassette). VSV-G, vesicular stomatitis virus G-protein.

Figure 3.7

Stable cell lines developed to generate lentiviral vectors. Transgene, envelope, and packaging cassettes introduced into vector’s producer cells by stable transfection following by selection. Tet-inducible system is commonly employed to circumvent the toxicity associated with the viral proteins. Tetracycline transactivator, ttA can be expressed from the same cassette (transgene cassette) as does a transgene.

Figure 3.8

Episomal forms generated during the HIV infection. Episomal genomes of the HIV-1 appear in four major forms: linear episomes: precursors for integration and for the rest of the episomes; 2-LTR (double-LTR) and 1-LTR (single-LTR), are aberrant circular forms, generated by the DNA repair machinery of the host, or through the aberrant reverse transcription reaction; the autointegrative forms are product of self-integration mediated by the host repair machinery. LTR, long terminal repeat.

Figure 3.9

HSV structure. HSV, Herpes simplex virus.

Figure 3.10

Tetracycline-controlled transcriptional activation. Dox = doxycycline, TA = transactivator, TetO = tetracycline operator.